Method for detecting the coupling rate of peg in coupling hyaluronic acid

Abstract

The application discloses a method for detecting the coupling rate of PEG in hyaluronic acid, which comprises the following steps: passing a sample to be detected through a liquid phase system, not connecting a chromatographic column in the liquid phase system, and directly entering a mass spectrometry system for detection; using a control solution; and quantifying by an external standard curve method. The detection method only needs 5 minutes for one needle operation, the liquid phase system is directly connected with the mass spectrometry system through two connectors, no chromatographic column is connected, and the problem that the sample is adsorbed in the chromatographic column and lost is avoided. The method is convenient to operate and high in accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis, in particular to a method for detecting the PEG coupling rate of coupled hyaluronic acid. BACKGROUND

[0002] Hyaluronic acid (HA) is a linear high-molecular-weight viscous polysaccharide composed of repeating disaccharide units of glucuronic acid and acetylglucosamine. Hyaluronic acid is an endogenous substance in the human body, has good biocompatibility, has high viscoelasticity and non-Newtonian rheological properties, is non-toxic, non-immunogenic and non-irritating, has high safety, and can be degraded and eliminated by injection of hyaluronidase, and is widely used as a soft tissue filler in beauty, which is injected into the skin to increase the volume of soft tissue, and can achieve the purpose of wrinkle removal or shaping.

[0003] The water-light needle of a medical beauty product is obtained by coupling reaction of a sodium hyaluronate solution and methoxy-polyethylene glycol-epoxide (M-EPOX) in an alkaline solution. The coupling rate of M-EPOX and sodium hyaluronate is an important property of the finished water-light needle product. The coupling function is to prolong the degradation time of the water-light needle product in the body, and the coupling rate will affect the molecular weight of the finished product. The molecular weight is an important parameter of the water-light needle product, reflecting the characteristics of the final product.

[0004] Patent document CN114397399A discloses a method for determining the content of quaternary ammonium salt in hyaluronic acid-quaternary ammonium salt polymer, which comprises: preparation of a test solution: hydrolyzing hyaluronic acid-quaternary ammonium salt polymer to obtain a mixed solution of hyaluronic acid and quaternary ammonium salt, then adding a salt solution and acetonitrile to precipitate the hyaluronic acid, and obtaining the supernatant as the test solution; using ion chromatography to determine the content of free quaternary ammonium salt in the test solution. In the above detection method, a carboxylic acid functionalized cation exchange chromatographic column is used to analyze the sample, which cannot avoid the defect of low detection accuracy caused by the adsorption of the sample in the chromatographic column.

[0005] Patent document CN108254456B discloses a method for determining the content of filled cross-linked dextran in sodium hyaluronate gel, which comprises the steps of high-temperature cracking analysis of a plurality of groups of pure cross-linked dextran with different masses by using cracking gas chromatography-mass spectrometry. In the above detection method, a polar chromatographic column is used to analyze the sample, which cannot avoid the defect of low detection accuracy caused by the adsorption of the sample in the chromatographic column.

[0006] At present, there is no clear method for testing the PEG coupling ratio of the water-light needle product, and the coupling ratio is a crucial factor for the quality of the water-light needle product. Therefore, it is urgent to establish a rapid and convenient method for detecting the PEG coupling ratio of the water-light needle product. SUMMARY

[0007] In order to overcome the deficiencies of the prior art, the present application aims to provide a method for detecting the direct coupling rate of the hyaluronic acid coupled PEG ratio, which can quickly and conveniently test the coupling ratio, thereby effectively controlling the product quality.

[0008] Glossary:

[0009] M-PEG-5K is methoxy-polyethylene glycol-5K;

[0010] M-EPOX is methoxy-polyethylene glycol-epoxide.

[0011] The present application provides, in a first aspect, a method for detecting the direct coupling rate of the hyaluronic acid coupled PEG ratio of a medical and cosmetic product, said detection method comprising the following steps:

[0012] (1) Standard curve: Take a control solution with a known content, dilute it with a solvent to obtain a linear control solution, inject the linear control solution into the liquid phase system (high performance liquid chromatograph), without connecting the chromatographic column, directly into the mass spectrometry system for detection, record the chromatogram, extract the PEG characteristic ion m / z:133.0859, obtain the control solution concentration, peak height, retention time, peak area and other parameters, and draw the standard curve of the linear relationship between the control solution and the peak height;

[0013] (2) Determination of coupling rate:

[0014] Dilute the sample to be tested (coupled hyaluronic acid to be tested) with water by a certain multiple, determine the peak height, and use the standard curve described in step (1) to obtain the PEG concentration c(PEG); determine the molecular weight M(hyaluronic acid or salt molecular weight) of hyaluronic acid or its salt;

[0015] The coupling rate is calculated by the following formulas ①②③

[0016] ①

[0017] ②

[0018] ③

[0019] Wherein, n(sample to be tested coupled PEG) is the amount of substance of the coupled PEG in the sample to be tested, c(PEG) is the concentration of the coupled PEG in the sample to be tested, V(sample to be tested volume) is the volume of the sample to be tested, n(hyaluronic acid or salt) is the amount of substance of hyaluronic acid or its salt, m(hyaluronic acid or salt input amount) is the mass of hyaluronic acid or its salt input, and M(hyaluronic acid or salt input amount) is the molecular weight of hyaluronic acid or its salt.

[0020] Further, the control solution is methoxypolyethylene glycol.

[0021] In one embodiment of the present application, the methoxypolyethylene glycol is selected from one of M-PEG-2K, M-PEG-5K, M-PEG-10K, M-PEG-20K and M-PEG-40K.

[0022] In one embodiment of the present application, the methoxypolyethylene glycol is M-PEG-5K.

[0023] Further, the molecular weight M of the hyaluronic acid or its salt (molecular weight of the hyaluronic acid or its salt) is determined by a multi-angle laser light scattering technique.

[0024] Further, the dilution fold in step (2) is 4000-20000 folds (such as 4000 folds, 4500 folds, 5000 folds, 5500 folds, 6000 folds, 6500 folds, 7000 folds, 7500 folds, 8000 folds, 8500 folds, 9000 folds, 9500 folds, 10000 folds, 15000 folds, 20000 folds), preferably 4000-10000 folds, more preferably 4000-8000 folds.

[0025] Further, the hyaluronic acid salt is selected from one or more of sodium salt, potassium salt, calcium salt, magnesium salt, zinc salt, cobalt salt and tetrabutylammonium salt of hyaluronic acid; preferably, the salt is sodium salt.

[0026] Further, the solvent in step (1) is water.

[0027] In one embodiment of the present application, the liquid phase system is SCIEX Exion LC liquid phase system.

[0028] In one embodiment of the present application, the mass spectrometry system is SCIEX TripleTOF6000 detector.

[0029] Further, the mobile phase in the liquid phase system is aqueous formic acid solution or aqueous trifluoroacetic acid solution.

[0030] In one embodiment of the present application, the mobile phase in the liquid phase system is aqueous formic acid solution.

[0031] Further, the mobile phase in the liquid phase system is a formic acid aqueous solution with a volume concentration of 0.01-0.5% (such as 0.01%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%), preferably, the mobile phase is a formic acid aqueous solution with a volume concentration of 0.1%.

[0032] Further, the column temperature in the liquid phase system is 25-45°C (such as 25, 28, 30, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45°C), preferably 38-42°C.

[0033] In an embodiment of the present application, the column temperature in the liquid phase system is 40°C.

[0034] Further, the flow rate in the liquid phase system is 0.05-1.0 mL / min (such as 0.05 mL / min, 0.1 mL / min, 0.15 mL / min, 0.16 mL / min, 0.17 mL / min, 0.18 mL / min, 0.19 mL / min, 0.2 mL / min, 0.21 mL / min, 0.22 mL / min, 0.23 mL / min, 0.24 mL / min, 0.25 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min), preferably, 0.05-0.5 mL / min, more preferably, 0.05-0.3 mL / min.

[0035] In an embodiment of the present application, the flow rate in the liquid phase system is 0.2 mL / min.

[0036] Further, the liquid phase system adopts an isocratic elution procedure.

[0037] Further, the elution time of the elution procedure is 1-10 min (such as 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min)

[0038] In an embodiment of the present application, the elution time of the elution procedure is 5 min.

[0039] Further, the sample injection volume in the liquid phase system is 1-10 μL (e.g., 1, 1.5, 2, 2.5, 3, 4, 5, 8, 9, 10 μL), preferably 1-8 μL.

[0040] In one embodiment of the present application, the sample injection volume in the liquid phase system is 2 μL.

[0041] Further, the parameters of the mass spectrometry system are as follows,

[0042] GAS1: 50, GAS2: 50, CUR: 30, TEM: 450, ISVF: 5500, DP: 50, CE: 40.

[0043] Further, the structure of the conjugated hyaluronic acid is as follows:

[0044] wherein n is the polymerization degree of the polyethylene glycol residue, and m is the polymerization degree of the hyaluronic acid residue, the molecular weight of the polyethylene glycol residue is 3000-8000 Da, and the molecular weight of the hyaluronic acid residue is 600-1500 kDa.

[0045] Further, the molecular weight of the polyethylene glycol residue is 4000-6000 Da (e.g., 4500 Da, 4600 Da, 4700 Da, 4800 Da, 4900 Da, 5000 Da, 5100 Da, 5200 Da, 5300 Da, 5400 Da, 5500 Da, 5600 Da, 5700 Da, 5800 Da, 5900 Da, 6000 Da).

[0046] Further, the molecular weight of the hyaluronic acid residue is 700-1300 kDa (e.g., 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1150 kDa, 1160 kDa, 1170 kDa, 1180 kDa, 1190 kDa, 1200 kDa, 1210 kDa, 1220 kDa, 1230 kDa, 1240 kDa, 1250 kDa, 1260 kDa, 1270 kDa, 1280 kDa, 1290 kDa, 1300 kDa), preferably 800-1200 kDa.

[0047] In one embodiment of the present application, the structure of the conjugated hyaluronic acid is as follows:

[0048]

[0049] In another aspect, the present application provides the use of the above-mentioned detection method in the quality evaluation of conjugated hyaluronic acid.

[0050] The present application has the following technical effects:

[0051] 1. The detection method of the present application is convenient and fast, the liquid phase system is directly connected with the mass spectrometry system through the two-way joint, the running time of one needle is only 5 minutes, the chromatographic column is not connected, and the problems of sample loss caused by adsorption of the sample in the chromatographic column are avoided;

[0052] 2. The search method of the present application is accurate and efficient, the qTOF is used for detection, the PEG characteristic ion m / z:133.0859 is extracted for quantification of PEG, the operation is convenient, and the accuracy is high. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is a linear relationship diagram of M-PEG-5K concentration and peak height. DETAILED DESCRIPTION

[0054] The technical solutions of the present application will be described in detail below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0055] Example 1

[0056] 1. Method parameters

[0057] Table 1

[0058]

[0059]

[0060] 2. Solution preparation

[0061] 2.1 Blank solution: Mili-Q water, taken fresh.

[0062] 2.2 Reference substance stock solution (20 mg / mL)

[0063] Weigh about 20 mg of M-PEG-5K sample into a centrifuge tube, add 1 mL of water to dissolve, and vortex to mix evenly.

[0064] 2.3 Intermediate working solution 1 (about 1 mg / mL)

[0065] Transfer 50 μL of 2.2 reference substance stock solution into a centrifuge tube, add 950 μL of water to dissolve, and vortex to mix evenly.

[0066] 2.4 Intermediate working solution 2 (about 100 μg / mL)

[0067] Transfer 100 μL of Intermediate Working Solution 1 in Section 2.3 into a centrifuge tube, add 900 μL water to dissolve, vortex to mix well.

[0068] 2.5 Intermediate Working Solution 3 (about 10 μg / mL)

[0069] Transfer 100 μL of Intermediate Working Solution 2 in Section 2.4 into a centrifuge tube, add 900 μL water to dissolve, vortex to mix well.

[0070] 2.6 Linear Solution Preparation

[0071] 2.6.1 Linear Solution 1 (about 0.2 μg / mL)

[0072] Transfer 20 μL of Intermediate Working Solution 3 in Section 2.5 into a centrifuge tube, add 980 μL water to dissolve, vortex to mix well. 2.6.2 Linear Solution 2 (about 0.5 μg / mL)

[0073] Transfer 50 μL of Intermediate Working Solution 3 in Section 2.5 into a centrifuge tube, add 950 μL water to dissolve, vortex to mix well. 2.6.3 Linear Solution 3 (about 1 μg / mL)

[0074] Transfer 100 μL of Intermediate Working Solution 3 in Section 2.5 into a centrifuge tube, add 900 μL water to dissolve, vortex to mix well.

[0075] 2.6.4 Linear Solution 4 (about 2 μg / mL)

[0076] Transfer 200 μL of Intermediate Working Solution 3 in Section 2.5 into a centrifuge tube, add 800 μL water to dissolve, vortex to mix well.

[0077] 2.6.5 Linear Solution 5 (about 5 μg / mL)

[0078] Transfer 500 μL of Intermediate Working Solution 3 in Section 2.5 into a centrifuge tube, add 500 μL water to dissolve, vortex to mix well.

[0079] 3. Linear Results

[0080] The PEG characteristic ion m / z: 133.0859 was extracted for quantitation of PEG in linear solutions. The standard curve was plotted with M-PEG-5K concentration as the horizontal coordinate and peak height as the vertical coordinate (see Figure 1 ).

[0081] Table 2

[0082] M-PEG-5K concentration (pg / mL) 0.21 0.52 1.03 2.07 5.16 Peak height 496.40 1516.39 2830.00 5706.06 12373.96

[0083] The results showed that M-PEG-5K was linearly compliant in the concentration range of 0.21-5.16 μg / mL and could be used for quantitative evaluation.

[0084] 4. Sample testing

[0085] 4.1 Preparation of sample solution:

[0086] Because the sample solution has a certain viscosity, in order to avoid clogging the sample needle, the sample solution is diluted with water by a certain multiple before sampling. It is found in the experiment that the sample has ionization matrix effect, and when the dilution multiple needs to be large enough, the concentration is positively correlated with the peak height. In this experiment, the results of diluting the finished product by 4000 times or more are used as the final calculation data.

[0087] 4.2 Calculation formula of coupling ratio:

[0088]

[0089]

[0090]

[0091] Among them, the concentration of PEG substance is obtained by substituting the peak height of the solution to be tested into the linear curve and using the external standard method. The molecular weight of sodium hyaluronate is directly measured by multi-angle light.

[0092] 4.3 Calculation results:

[0093]

[0094]

[0095] Through the experiment, the mol ratio of the input sodium hyaluronate to the PEG unit coupled can be calculated, which is provided to the personnel of the production department for the characterization of the ratio of PEG coupling on the water-light needle sample.

[0096] The first step of the experimental procedure for coupling PEG to sodium hyaluronate was detected using this method. The actual mass of PEG substance was calculated by measuring the concentration value of the solution, the volume value of the solution and the dilution multiple, and compared with the theoretical value (theoretical feeding amount). The test result meets the expected feeding result, and this method is accurate and feasible.

Claims

1. A method for detecting the coupling ratio of PEG to hyaluronic acid, characterized by, The detection method comprises the following steps: (1) Preparation of a standard curve: taking a control solution with a known content, diluting it with a solvent to obtain linear control solutions with different gradients; injecting the linear control solutions with different gradients into a liquid chromatography analysis system, not connecting a chromatographic column, and directly detecting a mass spectrometry system to record a chromatogram, and obtaining the concentration, peak height, retention time, and peak area of the control solution by extracting PEG characteristic ions m / z: 133.0859, and drawing a standard curve of the linear relationship between the control solution and the peak height; (2) Determination of the coupling rate: The sample to be tested is diluted with water to a certain multiple, and the peak height is measured, DP: 50, CE: 40, m / z: 133.0859, and the PEG concentration c is obtained by using the standard curve described in step (1) PEG ; The molecular weight M of hyaluronic acid or its salt is measured 透明质酸或其盐分子量 ; The coupling rate is calculated by the following formulas ①, ②, and ③ wherein n 待测样品中偶联PEG is the amount of substance of the substance coupled with PEG in the sample to be measured, mol, c PEG is the concentration of the substance coupled with PEG in the sample to be measured, V 待测样品体积 is the volume of the sample to be measured, n 透明质酸或其盐 is the amount of substance of the hyaluronic acid or salt thereof, m 透明质酸或其盐投入量 is the mass of the hyaluronic acid or salt thereof introduced, mol, M 透明质酸或其盐投入量 is the molecular weight of the hyaluronic acid or salt thereof; The mobile phase in the liquid phase system is a formic acid aqueous solution or a trifluoroacetic acid aqueous solution; The liquid phase system adopts an isocratic elution program, and the flow rate in the liquid phase system is 0.05-1.0 mL / min; The dilution multiple in step (2) is 4000-20000 times; The structure of the coupled hyaluronic acid is: wherein n is the degree of polymerization of the polyethylene glycol residue, m is the degree of polymerization of the hyaluronic acid residue, the molecular weight of the polyethylene glycol residue is 3000-8000 Da, and the molecular weight of the hyaluronic acid residue is 600-1500 kDa.

2. The detection method of claim 1, wherein, The molecular weight M of the hyaluronic acid or salt thereof 透明质酸或其盐分子量 The method for measuring the molecular weight M is the multi-angle laser light scattering technique.

3. The detection method as described in claim 1, characterized in that, The control solution is methoxypolyethylene glycol, and the methoxypolyethylene glycol is selected from one of M-PEG-2K, M-PEG-5K, M-PEG-10K, M-PEG-20K, and M-PEG-40K.

4. The detection method as described in claim 1, characterized in that, The mobile phase in the liquid phase system is a formic acid aqueous solution.

5. The detection method as described in claim 2, characterized in that, The mobile phase in the liquid phase system is a formic acid aqueous solution with a volume concentration of 0.01-0.5%.

6. The detection method as described in claim 2, characterized in that, The mobile phase is a formic acid aqueous solution with a volume concentration of 0.1%.

7. The detection method as described in claim 1, characterized in that, The dilution multiple in step (2) is 4000-10000 times.

8. The method of claim 1, wherein, The dilution multiple in step (2) is 4000-8000 times.

9. The method of claim 1, wherein, The hyaluronic acid salt is selected from one or more of sodium salt, potassium salt, calcium salt, magnesium salt, zinc salt, cobalt salt, and tetrabutylammonium salt of hyaluronic acid.

10. The detection method of claim 9, wherein, The hyaluronic acid salt is a sodium salt.

11. The method of claim 1, wherein, The flow rate in the liquid phase system is 0.05-0.5 mL / min.

12. The method of claim 1, wherein, The flow rate in the liquid phase system is 0.05-0.3 mL / min.

13. The detection method as described in claim 1, characterized in that, The molecular weight of the polyethylene glycol residue is 4000-6000 Da, and the molecular weight of the hyaluronic acid residue is 700-1300 kDa.

14. Use of the detection method according to any one of claims 1-13 in the evaluation of the quality of coupled hyaluronic acid.

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