Method for detecting content of multi-component leached substances in intraocular lens by liquid chromatography

Abstract

The invention discloses a method for detecting the content of multi-component leached substances of an intraocular lens by liquid chromatography. The method comprises the following steps: preparing a series of standard solutions with different concentrations of reference substances of the leached substances of the intraocular lens; carrying out gradient elution on the standard solutions of the reference substances of the leached substances by adopting liquid chromatography to obtain chromatograms of the leached substances corresponding to different retention times, and drawing a standard curve of each standard solution according to the concentration-peak area; the method comprises the following steps: adding an intraocular lens sample to be detected into an organic solvent, and carrying out simulated extraction in a water bath kettle to obtain a test solution of a multi-component leached substance of the sample; performing liquid chromatography detection on the test solution to obtain a sample chromatogram; and according to a sample chromatogram and a result obtained by the standard solution, carrying out qualitative and quantitative analysis on the leached substance components in the test solution by using an external standard method. Qualitative and quantitative operation of multiple components can be completed at a time, chromatographic separation and linear relation are good, precision and accuracy are high, operation is easy and convenient, and rapidness and high efficiency are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for detecting the content of multi-component leachable substances in an artificial lens by using a liquid chromatography method. Background Art

[0002] Intraocular lens, also known as IOL, refers to a special lens made of artificial synthetic materials. It is mainly used to replace the diseased lens in cataract surgery and is one of the most widely used implantable medical device products in the world. With the aging of the population, the number of patients undergoing cataract removal combined with intraocular lens implantation has also increased year by year. As the user population continues to expand, in order to meet the specific needs of different patients, intraocular lens materials have shown diversified development, mainly including silicone gel materials with high thermal stability, hydrophobic acrylic materials with long-term stability and high mechanical strength, and hydrophilic acrylic materials with good hydrophilicity and easy folding. Strong stability, high optical quality and excellent biocompatibility are the fundamental guarantees for improving patients' postoperative visual quality, and biocompatibility is more important than the other two. The safety of intraocular lens manufacturing materials is closely related to the user's eye health and has received special attention.

[0003] At present, the industry standard "YY / T 0290.5-2023-Ophthalmic Optics Intraocular Lenses Part 5: Biocompatibility" and the national standard "GB / T 16886.17-2005-Biological Evaluation of Medical Devices Part 17 Establishment of Permissible Limits of Leachables" stipulate the safety evaluation of leachables in intraocular lenses and require the establishment of safety limits for contact with the human body. The leachable content of medical devices is one of the important indicators for evaluating biocompatibility and has also been a focus of attention in the review process of medical devices in recent years. Therefore, the analysis of the residual amount of leachable components in intraocular lenses is a prerequisite for the safety study of intraocular lenses. It also helps to evaluate intraocular lens materials and determine preclinical inspection procedures. It is a key requirement for the production and development of intraocular lenses.

[0004] The current IOL standards lack a clear method for detecting leachables, which poses a major challenge to product safety supervision. Although some scholars have conducted research, there are few reports and they mainly focus on testing a single component, which takes a long time to test. In particular, research on multi-component leachables is very scarce. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for detecting the content of multi-component leachable substances in artificial lenses by liquid chromatography, which can simultaneously detect the content of multi-component leachable substances in artificial lenses based on liquid chromatography ultraviolet method, and the method is simple, rapid and highly sensitive.

[0006] The present invention discloses a method for detecting the content of multi-component leachable substances in an intraocular lens by liquid chromatography, comprising:

[0007] Step 1, weighing the intraocular lens leachable reference substances respectively, and then preparing a series of standard solutions of different concentrations for each leachable reference substance with an organic solvent;

[0008] Step 2: gradient eluting the series of standard solutions of each leachable reference substance prepared in step 1 by liquid chromatography to obtain leachable chromatograms corresponding to different retention times, and drawing a standard curve of each standard solution according to concentration-peak area;

[0009] Step 3, adding the artificial lens sample to be tested into an organic solvent, and placing it in a water bath for simulated leaching to obtain a test solution of a multi-component leachable substance of the artificial lens sample to be tested;

[0010] Step 4: Perform liquid chromatography on the test solution obtained in step 3 to obtain a sample chromatogram; and perform qualitative and quantitative analysis of the leachable components in the test solution using an external standard method based on the sample chromatogram and the result obtained in step 2.

[0011] As a further improvement of the present invention, the leachable substances of the intraocular lens include 6 or more of 2-hydroxyethyl methacrylate (HEMA), 2-ethoxyethyl methacrylate (EOEMA), ethylene glycol dimethacrylate (EGDMA) and trimethylolpropane trimethacrylate (TMPTMA), methyl methacrylate (MMA), hydroxyethyl acrylate (HEA), glyceryl methacrylate (GMA) and lauryl methacrylate (LMA).

[0012] As a further improvement of the present invention, in step 1, the preparation method of the multi-component mixed standard solution is:

[0013] A mixed standard solution containing a target detection object reference substance is prepared with an organic solvent, and the mixed standard solution is diluted stepwise with the organic solvent to obtain a series of standard solutions with concentrations ranging from 1 to 50 μg / mL.

[0014] As a further improvement of the present invention, in step 1, the leachable material reference substance is 6 or more of the following: 2-hydroxyethyl methacrylate (HEMA) reference substance, 2-ethoxyethyl methacrylate (EOEMA) reference substance, ethylene glycol dimethacrylate (EGDMA) reference substance, trimethylolpropane trimethacrylate (TMPTMA) reference substance, methyl methacrylate (MMA) reference substance, hydroxyethyl acrylate (HEA) reference substance, glyceryl methacrylate (GMA) reference substance and lauryl methacrylate (LMA) reference substance; and the organic solvent is one or more of methanol, ethanol, isopropanol, acetonitrile, acetone, n-hexane and dichloromethane.

[0015] As a further improvement of the present invention, in step 2, the test conditions of the liquid chromatography are:

[0016] The chromatographic column is alkyl silica gel; mobile phase A is water, and mobile phase B is acetonitrile; the column temperature is 20-50°C; the flow rate is 0.7-1.5 ml / min; the detector is a DAD detector or a UV detector; the detection wavelength is 200-400 nm; and the injection volume is 1-20 μL.

[0017] As a further improvement of the present invention, in step 2, the gradient elution procedure is: from 0 to 1 min, the volume fraction of mobile phase B is 5%; from 1 to 11 min, the volume fraction of mobile phase B is increased from 5% to 100% and maintained for 6 min; from 17 to 18 min, the volume fraction of mobile phase B is reduced from 100% to 5%.

[0018] As a further improvement of the present invention, the chromatographic column filler is octadecyl, octyl, phenyl or adamantyl, preferably CAPCELL PAK C18, CAPCELL PAK ADME or other equivalent chromatographic columns.

[0019] As a further improvement of the present invention, in step 3, the simulated extraction method is: add 1 g of the artificial lens sample to be tested into 10-20 ml of an organic solvent, place the mixture in a 35±2°C water bath and culture it for 72 h±1 h for simulated extraction to obtain a test solution of a multi-component leachable substance of the artificial lens sample to be tested.

[0020] As a further improvement of the present invention, in step 4, the method of using the external standard method to qualitatively and quantitatively analyze the leachable components in the test solution is as follows: comparing the retention time of each peak in the sample chromatogram with the leachable chromatogram in step 2 to determine the type of leachable corresponding to each peak; and then calculating the content of the leachable in the test solution based on the peak area and the standard curve in step 2.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention adopts high performance liquid chromatography for gradient elution, and qualitatively and quantitatively analyzes multi-component leachable substances by an external standard method; that is, firstly, a reference solution is detected to determine the types of leachable substances corresponding to each retention time, a series of concentrations of a multi-component mixed standard solution is prepared, and a relationship curve (i.e., a standard curve) between the concentration of the leachable substances and the peak area is obtained; finally, a test solution is detected, and the leachable substance components in the test solution are qualitatively and quantitatively analyzed by an external standard method in combination with the obtained spectrum, the retention time of the reference substance and the standard curve; the method of the invention can complete the qualitative and quantitative analysis of multiple components at one time, and has the characteristics of being simple, rapid, efficient, sensitive and accurate.

[0023] By optimizing the test conditions, the present invention can not only detect the content of multi-component leachables at the same time to ensure the safety of clinical implantation of artificial lenses, but also improve the detection efficiency, comply with the technical review guidelines for verification and confirmation of registration of known leachables determination methods for medical devices, and the linearity, quantitative limit, detection limit, precision, accuracy, etc. meet the requirements of methodological verification; the method has been applied to the detection and analysis of the content of leachables in artificial lens products, and can be widely used in the supervision of the content of leachables in artificial lens products and product quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention discloses a flow chart of a method for detecting the content of multi-component leachable substances in an intraocular lens by liquid chromatography;

[0025] Figure 2 It is a liquid chromatogram obtained from the multi-component mixed standard solution in Example 1 of the present invention; wherein 1 is glyceryl methacrylate (GMA), 2 is 2-hydroxyethyl methacrylate (HEMA), 3 is 2-ethoxyethyl methacrylate (EOEMA), 4 is ethylene glycol dimethacrylate (EGDMA), 5 is trimethylolpropane trimethacrylate (TMPTMA), and 6 is lauryl methacrylate (LMA);

[0026] Figure 3 This is the liquid chromatogram obtained from the test solution in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0029] like Figure 1 As shown, the present invention provides a method for detecting the content of multi-component leachables of an intraocular lens by liquid chromatography, which can complete the qualitative and quantitative detection of multi-component leachables of an intraocular lens at one time, and the leachables of the intraocular lens in the intraocular lens include 6 or more of 2-hydroxyethyl methacrylate (HEMA), 2-ethoxyethyl methacrylate (EOEMA), ethylene glycol dimethacrylate (EGDMA), trimethylolpropane trimethacrylate (TMPTMA), methyl methacrylate (MMA), hydroxyethyl acrylate (HEA), glyceryl methacrylate (GMA) and lauryl methacrylate (LMA); the method comprises:

[0030] Step 1, weigh the artificial lens leachable reference substance respectively, prepare a mixed standard solution containing the target detection substance reference substance with an organic solvent, and dilute the mixed standard solution with the organic solvent step by step to obtain a series of standard solutions with a concentration range of 1 to 50 μg / mL; wherein the leachable reference substance is 6 or more of the following: 2-hydroxyethyl methacrylate (HEMA) reference substance, 2-ethoxyethyl methacrylate (EOEMA) reference substance, ethylene glycol dimethacrylate (EGDMA) reference substance, trimethylolpropane trimethacrylate (TMPTMA) reference substance, methyl methacrylate (MMA) reference substance, hydroxyethyl acrylate (HEA) reference substance, glyceryl methacrylate (GMA) reference substance, and lauryl methacrylate (LMA) reference substance; the organic solvent is one or more of methanol, ethanol, isopropanol, acetonitrile, acetone, n-hexane, and dichloromethane;

[0031] Step 2: gradient eluting the series of standard solutions of each leachable reference substance prepared in step 1 by liquid chromatography to obtain leachable chromatograms corresponding to different retention times, and drawing a standard curve of each standard solution according to concentration-peak area; wherein,

[0032] The test conditions of liquid chromatography are:

[0033] The chromatographic column is alkyl silica gel; mobile phase A is water, and mobile phase B is acetonitrile; the column temperature is 20-50° C.; the flow rate is 0.7-1.5 ml / min; the detector is a DAD detector or a UV detector; the detection wavelength is 200-400 nm; the injection volume is 1-20 μL; further, the chromatographic column filler is octadecyl, octyl, phenyl or adamantyl, preferably CAPCELL PAK C18, CAPCELLPAK ADME or other equivalent chromatographic columns;

[0034] The procedure of gradient elution is as follows: from 0 to 1 minute, the volume fraction of mobile phase B is 5%; from 1 to 11 minutes, the volume fraction of mobile phase B increases from 5% to 100% and is maintained for 6 minutes; from 17 to 18 minutes, the volume fraction of mobile phase B decreases from 100% to 5%.

[0035] Step 3, adding 1 g of the artificial lens sample to be tested into 10-20 ml of an organic solvent, placing the mixture in a 35±2° C. water bath and incubating it for 72 h±1 h for simulated leaching to obtain a test solution of a multi-component leachable substance of the artificial lens sample to be tested;

[0036] Step 4: Perform liquid chromatography on the test solution obtained in step 3 using the same adjustment as step 2 to obtain a sample chromatogram; according to the sample chromatogram and the results obtained in step 2 (chromatogram and standard curve of the mixed standard solution), the leachable components in the test solution are qualitatively and quantitatively determined using the external standard method; wherein the specific qualitative and quantitative methods are:

[0037] Compare the retention time of each peak in the sample chromatogram with the leachable substance chromatogram in step 2 to determine the type of leachable substance corresponding to each peak; then calculate the content of leachable substance in the test solution based on the peak area and the standard curve in step 2.

[0038] Example 1

[0039] like Figure 1 As shown, the present invention provides a method for detecting the content of multi-component leachable substances in an intraocular lens by liquid chromatography, comprising:

[0040] S1. Prepare standard solution of intraocular lens leachable material:

[0041] Use a 1 / 100,000 balance to accurately weigh the artificial lens leachable material reference substances: 0.1 g each of GMA, HEMA, EOEMA, EGDMA, TMPTMA and LMA, add them to 4 100 ml volumetric flasks respectively, dissolve and make up the volume with acetonitrile to prepare a mother solution with a concentration of 1000 μg / ml; then take 1 ml of the above mother solution respectively in 1 10 ml volumetric flask, make up the volume with acetonitrile to prepare a mixed standard solution with a concentration of 100 μg / ml; then further dilute each artificial lens leachable material step by step according to the same method to prepare a gradient mixed standard solution with concentrations of 1 μg / ml, 2 μg / ml, 5 μg / ml, 10 μg / ml, 30 μg / ml and 50 μg / ml.

[0042] S2. Draw a standard curve for intraocular lens leachables:

[0043] The standard solution was subjected to gradient elution by high performance liquid chromatography, wherein the chromatographic column was CAPCELL PAKADME (4.6mm*250mm*5μm); mobile phase A was water, and mobile phase B was acetonitrile; the column temperature was 30°C; detector: UV detector; detection wavelength was 210nm; injection volume was 10μL. The program for mobile phase gradient elution was set as follows: from 0 to 1 min, the volume fraction of mobile phase B was 5%; from 1 to 11 min, the volume fraction of mobile phase B increased from 5% to 100% and maintained for 6 min; from 17 to 18 min, the volume fraction of mobile phase B decreased from 100% to 5%, and the flow rate was maintained at 0.7 ml / min. After the above gradient elution, the following was obtained: Figure 2 Chromatogram of the standard solution is shown.

[0044] The detection limit (LOD, LOD = 3S / N) of the method was three times the signal-to-noise ratio S / N, and the quantification limit (LOQ, LOQ = 10S / N) of the method was ten times the signal-to-noise ratio S / N. The detection limit and quantification limit of each leachable substance in acetonitrile were calculated in combination with the addition of matrix. The relevant parameters of the standard curve of each leachable substance based on the above matrix, LOD and LOQ related information are shown in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] Depend on Figure 2 As can be seen from Table 1, the method of the present invention can achieve rapid detection of multi-component leachables within 24 minutes with high detection sensitivity. The peak area and concentration show a good linear relationship in the respective concentration ranges, and the linear correlation coefficient R is not less than 0.99. The detection limit of the method is 0.08-0.20 μg / ml.

[0049] S3. Precision and accuracy test:

[0050] Repeatability was used to examine precision, and recovery was used to evaluate the accuracy of the method. Standard solutions were precisely added to blank matrices that did not contain the components to be tested to obtain spiked samples at three different concentration levels (low, medium, and high, see actual concentration for details). Each concentration level was repeated 3 times, and the recovery and relative standard deviation (RSD) values ​​were calculated. The results are shown in Table 2.

[0051] Table 2

[0052]

[0053]

[0054] According to Table 2, the relative standard deviation is <3%, indicating that the test method has good repeatability and high precision. Through the spike test, the recovery rates of the six leachables were between 90% and 105%, indicating that the test method has high accuracy.

[0055] S4. Prepare the test solution of the intraocular lens sample to be tested:

[0056] Take 1 g of intraocular lens sample, add 10 ml of acetonitrile solvent, place it in a 35±2°C water bath and culture it for 72 h±1 h for simulated leaching to obtain a test solution of intraocular lens multi-component leachable substances.

[0057] S5. Sample testing:

[0058] 10 μL of the test solution obtained in S4 was directly injected into the liquid chromatograph and tested according to the above instrument conditions. The type of leachable substance corresponding to each peak was obtained by comparing the retention time with the reference substance, and then the content of leachable substance of each component was calculated according to the fitting curve (i.e., standard curve) of each leachable substance reference substance. The results are as follows: Figure 3 As shown. The test results show that the leachable substances in the sample to be tested are HEMA, EOEMA and EGDMA, with contents of 25.1μg / ml, 40.0μg / ml and 2.7μg / ml respectively, and the spiked recoveries are 103.5%, 102.5% and 98.7% respectively, with relative standard deviations of 1.5%, 2.2% and 1.8%. It can be seen that the method of the present invention has high detection efficiency, good repeatability, accuracy and reliability.

[0059] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. 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 content of multi-component leachable substances in an intraocular lens by liquid chromatography, characterized in that: include: Step 1, weighing the intraocular lens leachable reference substances respectively, and then preparing a series of standard solutions of different concentrations for each leachable reference substance with an organic solvent; Step 2: gradient eluting the series of standard solutions of each leachable reference substance prepared in step 1 by liquid chromatography to obtain leachable chromatograms corresponding to different retention times, and drawing a standard curve of each standard solution according to concentration-peak area; Step 3, adding the artificial lens sample to be tested into an organic solvent, and placing it in a water bath for simulated leaching to obtain a test solution of a multi-component leachable substance of the artificial lens sample to be tested; Step 4: Perform liquid chromatography on the test solution obtained in step 3 to obtain a sample chromatogram; and perform qualitative and quantitative analysis of the leachable components in the test solution using an external standard method based on the sample chromatogram and the result obtained in step 2.

2. The method according to claim 1, characterized in that The leachable substances of the intraocular lens include 6 or more of 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, methyl methacrylate, hydroxyethyl acrylate, glyceryl methacrylate and lauryl methacrylate.

3. The method according to claim 1, characterized in that In step 1, the preparation method of the multi-component mixed standard solution is: A mixed standard solution containing a target detection object reference substance is prepared with an organic solvent, and the mixed standard solution is diluted stepwise with the organic solvent to obtain a series of standard solutions with concentrations ranging from 1 to 50 μg / mL.

4. The method according to claim 1, characterized in that In the step 1, the leachable material reference substance is 6 or more of 2-hydroxyethyl methacrylate reference substance, 2-ethoxyethyl methacrylate reference substance, ethylene glycol dimethacrylate reference substance, trimethylolpropane trimethacrylate reference substance, methyl methacrylate reference substance, hydroxyethyl acrylate reference substance, glyceryl methacrylate reference substance and lauryl methacrylate reference substance; the organic solvent is one or more of methanol, ethanol, isopropanol, acetonitrile, acetone, n-hexane and dichloromethane.

5. The method according to claim 1, characterized in that In step 2, the test conditions of the liquid chromatography are: The chromatographic column is alkyl silica gel; mobile phase A is water, mobile phase B is acetonitrile; column temperature is 20-50°C; flow rate is 0.7-1.5 ml / min; Detector: DAD detector or UV detector; detection wavelength is 200-400nm; injection volume is 1-20μL.

6. The method according to claim 5, characterized in that In step 2, the gradient elution procedure is as follows: from 0 to 1 minute, the volume fraction of mobile phase B is 5%; from 1 to 11 minutes, the volume fraction of mobile phase B increases from 5% to 100% and is maintained for 6 minutes; from 17 to 18 minutes, the volume fraction of mobile phase B decreases from 100% to 5%.

7. The method according to claim 5, characterized in that The chromatographic column filler is octadecyl, octyl, phenyl or adamantyl, preferably CAPCELL PAK C18, CAPCELL PAK ADME or other equivalent chromatographic columns.

8. The method according to claim 1, characterized in that In step 3, the simulated leaching method is: add 1 g of the artificial lens sample to be tested into 10-20 ml of an organic solvent, place it in a 35±2°C water bath and culture it for 72h±1h for simulated leaching to obtain a test solution of a multi-component leachable substance of the artificial lens sample to be tested.

9. The method according to claim 1, characterized in that: In step 4, the method of using the external standard method to qualitatively and quantitatively analyze the leachable components in the test solution is as follows: comparing the retention time of each peak in the sample chromatogram with the leachable chromatogram in step 2 to determine the type of leachable corresponding to each peak; and then calculating the content of the leachable in the test solution based on the peak area and the standard curve in step 2.

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