Method for detecting multi-component leachables content of intraocular lenses by liquid chromatography

Abstract

The application discloses a method for detecting the content of multi-component leachable of intraocular lens by liquid chromatography, comprising the following steps: preparing a series of standard solutions with different concentrations of the leachable of intraocular lens; performing gradient elution on the standard solutions of the leachable of intraocular lens by liquid chromatography to obtain chromatograms of the leachable corresponding to different retention times, and drawing standard curves of the standard solutions according to the concentration-peak area; adding the intraocular lens sample to be detected into an organic solvent and placing the sample in a water bath to simulate extraction to obtain a test sample solution of the multi-component leachable of the sample; performing liquid chromatography on the test sample solution to obtain a sample chromatogram; and using an external standard method to qualitatively and quantitatively analyze the leachable components in the test sample solution according to the sample chromatogram and the results obtained by the standard solutions. The method can qualitatively and quantitatively analyze multiple components at one time, has good chromatographic separation and linear relationship, high precision and accuracy, and is simple, rapid and efficient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of analytical detection, and particularly relates to a method for detecting the content of multi-component leachable substances of an intraocular lens by liquid chromatography. BACKGROUND

[0002] An intraocular lens, also known as an IOL, is a special lens made of synthetic materials, mainly used to replace the diseased lens in cataract surgery, and is one of the most widely used implantable medical devices in the world. With the increasing aging of the population, the number of patients undergoing cataract extraction combined with intraocular lens implantation is also increasing 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 acrylate materials with long-term stability and high mechanical strength, and hydrophilic acrylate materials with good hydrophilicity and easy folding. Strong stability, high optical quality and excellent biocompatibility are the fundamental guarantee for improving the postoperative visual quality of patients, and biocompatibility is more important than the other two, and the safety of intraocular lens manufacturing materials is closely related to the eye health of the user and receives special attention.

[0003] Currently, 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 allowable limit of leachable substances have specified the safety evaluation of leachable substances of intraocular lenses, and require to establish a safety limit for contact with the human body. The content of leachable substances of medical devices is one of the important indicators for evaluating biocompatibility, and is also the focus of attention in the medical device review process in recent years. Therefore, the analysis of the residual amount of leachable components of intraocular lenses is the premise of the safety research of intraocular lenses, and also helps to evaluate the intraocular lens materials and helps to determine the preclinical examination procedures, which is the key requirement for the development of intraocular lenses.

[0004] The current intraocular lens standard still lacks a clear method for detecting leachable substances, which brings great difficulties to the safety supervision of products. Although some scholars have conducted research, there are few reports and the tests are mainly for single components, the detection time is long, and especially the research on multi-component leachable substances is very scarce. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a method for detecting the content of multi-component leachable substances of an intraocular lens by liquid chromatography, which can simultaneously detect the content of multi-component leachable substances of an intraocular lens based on liquid chromatography ultraviolet method, and the method is simple, rapid and high in sensitivity.

[0006] The application discloses a method for detecting content of multi-component leachable substances of intraocular lenses by liquid chromatography, comprising the following steps:

[0007] In step 1, the leachable substance control samples of the intraocular lenses are weighed respectively, and then a series of standard solutions with different concentrations are prepared for each leachable substance control sample by using an organic solvent.

[0008] In step 2, the series of standard solutions of each leachable substance control sample prepared in step 1 are subjected to gradient elution by using liquid chromatography, so that leachable substance chromatograms corresponding to different retention times are obtained, and a standard curve of each standard solution is drawn according to the concentration-peak area.

[0009] In step 3, the intraocular lens sample to be detected is added into an organic solvent and placed in a water bath to simulate extraction, so that a test sample solution of multi-component leachable substances of the intraocular lens sample to be detected is obtained.

[0010] In step 4, the test sample solution obtained in step 3 is subjected to liquid chromatography detection, so that a sample chromatogram is obtained; according to the sample chromatogram and the result obtained in step 2, the leachable substance components in the test sample solution are subjected to qualitative and quantitative determination by using an external standard method.

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

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

[0013] The mixed standard solution containing the target detection control sample is prepared by using an organic solvent, and the mixed standard solution is diluted by using the organic solvent step by step, so that a series of standard solutions with a concentration in the range of 1-50 μg / mL are obtained.

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

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

[0016] The chromatographic column is alkyl silica gel; the mobile phase A is water, and the 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 application, in the step 2, the gradient elution program is as follows: 0-1 min, the volume fraction of the mobile phase B is 5%; 1-11 min, the volume fraction of the mobile phase B is increased from 5% to 100% and maintained for 6 min; and 17-18 min, the volume fraction of the mobile phase B is decreased from 100% to 5%.

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

[0019] As a further improvement of the present application, in the step 3, the method of simulating the extraction is as follows: 1 g of the intraocular lens sample to be tested is added to 10-20 ml of an organic solvent, and placed in a 35±2°C water bath for 72 h±1 h to simulate the extraction, thereby obtaining a test sample solution of the multi-component leachable of the intraocular lens sample to be tested.

[0020] As a further improvement of the present application, in the step 4, the method of using an external standard for the qualitative and quantitative determination of the leachable components in the test sample solution is as follows: the retention time of each peak in the sample chromatogram is compared with the leachable chromatogram in the step 2 to determine the leachable species corresponding to each peak; and then the content of the leachable in the test sample solution is calculated according to the peak area and the standard curve in the step 2.

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

[0022] The present application adopts high performance liquid chromatography for gradient elution, and the multi-component leachable is qualitatively and quantitatively determined by an external standard method; that is, first, the control solution is detected to determine the leachable types corresponding to each retention time, a multi-component mixed standard solution series concentration is prepared, the relationship curve (i.e., the standard curve) between the leachable concentration and the peak area is obtained, finally, the test solution is detected, the obtained spectrum, the retention time of the control and the standard curve are combined, and the leachable components in the test solution are qualitatively and quantitatively determined by the external standard method; the method of the present application can qualitatively and quantitatively determine multiple components at one time, and has the characteristics of simplicity, rapidness, high efficiency, sensitivity and accuracy.

[0023] The present application can not only simultaneously detect the content of multi-component leachable, ensure the safety of intraocular lens clinical implantation, but also improve the detection efficiency, meet the known leachable determination method verification and confirmation registration technical review guidelines of medical devices, and meet the requirements of methodological verification in terms of linearity, quantitative limit, detection limit, precision and accuracy; the method has been applied to the detection and analysis of the leachable content in intraocular lens products, and can be widely applied to the supervision and product quality control of the leachable content in intraocular lens products. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The flowchart of the method for detecting the multi-component leachable content of intraocular lens by liquid chromatography disclosed in the present application is shown in the figure;

[0025] Figure 2 The liquid chromatogram obtained by the multi-component mixed standard solution in Example 1 of the present application is shown in the figure; 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 The liquid chromatogram obtained by the test solution in Example 1 of the present application is shown in the figure. DETAILED DESCRIPTION

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The present invention will now be described in further detail with reference to the accompanying drawings:

[0029] like Figure 1 As shown, this invention provides a method for detecting the content of multiple leachable components in an intraocular lens (IOL) using liquid chromatography. This method can perform qualitative and quantitative detection of multiple leachable components in an IOL in a single step. The leachable components in the IOL include six or more of the following: 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 includes:

[0030] Step 1: Weigh out the intraocular lens leachable reference standards separately, and prepare a mixed standard solution containing the target analyte reference standard using an organic solvent. Dilute the mixed standard solution stepwise with the organic solvent to obtain a series of standard solutions with concentrations ranging from 1 to 50 μg / mL. The leachable reference standards are six or more of the following: 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 organic solvent is one or more of the following: methanol, ethanol, isopropanol, acetonitrile, acetone, n-hexane, and dichloromethane.

[0031] Step 2: Gradient elution of the series of standard solutions of each leachable reference standard prepared in Step 1 was performed using high-performance liquid chromatography (HPLC) to obtain chromatograms of the leachable substances corresponding to different retention times. A standard curve was then plotted for each standard solution based on the concentration-peak area relationship.

[0032] The test conditions for liquid chromatography are as follows:

[0033] The chromatographic column is alkyl silica gel; the mobile phase A is water, and the mobile phase B is acetonitrile; the column temperature is 20-50 DEG 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 amount is 1-20 muL; further, the chromatographic column filler is octadecyl, octyl, phenyl or adamantyl, preferably CAPCELL PAK C18, CAPCELL PAK ADME or other equivalent chromatographic columns;

[0034] The gradient elution program is as follows: 0-1 min, the volume fraction of mobile phase B is 5%; 1-11 min, the volume fraction of mobile phase B is increased from 5% to 100%, and kept for 6 min; 17-18 min, the volume fraction of mobile phase B is decreased from 100% to 5%.

[0035] Step 3, 1g of the artificial lens sample to be tested is added into 10-20 ml of an organic solvent, and is cultured in a 35±2 DEG C water bath for 72h±1h to simulate leaching, so as to obtain a test sample solution of the multi-component leachable of the artificial lens sample to be tested;

[0036] Step 4, the test sample solution obtained in step 3 is subjected to the same adjustment as in step 2 for liquid chromatography detection, so as to obtain a sample chromatogram; according to the sample chromatogram and the results (chromatogram of the mixed standard solution and the standard curve) obtained in step 2, the external standard method is used to qualitatively and quantitatively determine the leachable components in the test sample solution; wherein, the specific qualitative and quantitative method is as follows:

[0037] The retention time of each peak in the sample chromatogram is compared with the leachable chromatogram in step 2, so as to determine the leachable types corresponding to each peak; then, the content of the leachable in the test sample solution is calculated according to the peak area and the standard curve in step 2.

[0038] Example 1

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

[0040] S1, preparing a standard solution of artificial lens leachable:

[0041] Accurately weigh the IOL leachable control samples: GMA, HEMA, EOEMA, EGDMA, TMPTMA and LMA each 0.1 g, respectively add into 4 100 ml volumetric flasks, dissolve and dilute to volume with acetonitrile, prepare the mother liquor with a concentration of 1000 μg / ml; then take 1 ml of each of the above mother liquor into a 10 ml volumetric flask, dilute to volume with acetonitrile, prepare the mixed standard solution with a concentration of 100 μg / ml; then further dilute each IOL leachable to the gradient mixed standard solution with a concentration of 1 μg / ml, 2 μg / ml, 5 μg / ml, 10 μg / ml, 30 μg / ml, 50 μg / ml according to the same method.

[0042] S2, draw the standard curve of IOL leachable:

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

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

[0045] Table 1

[0046]

[0047]

[0048] It can be seen from Figure 2 and Table 1 that the method of the present application can realize the simultaneous rapid detection of multi-component leachable within 24 min, and has high detection sensitivity. The peak area and the concentration in the respective concentration range have a good linear relationship, and the linear correlation coefficient R is not less than 0.99, and the detection limit of the method is 0.08-0.20 μg / ml.

[0049] S3, Precision and accuracy test:

[0050] The repeatability is used to investigate the precision, and the recovery rate is used to evaluate the accuracy. The standard solution is precisely added to the blank matrix without the to-be-tested component to obtain three different concentration levels (low, medium and high, see actual concentration for details) of the spiked sample, each concentration level is repeated for 3 times, and the recovery rate and relative standard deviation RSD value are calculated, and the results are shown in Table 2.

[0051] Table 2

[0052]

[0053]

[0054] As can be seen from Table 2, the relative standard deviation is less than 3%, which indicates that the test method has good repeatability and high precision. Through the standard addition test, the recovery rate of the six leachable substances is between 90% and 105%, and it can be seen that the test method has high accuracy.

[0055] S4, Preparation of test solution of to-be-tested intraocular lens sample:

[0056] 1g of the intraocular lens sample is added into 10ml of acetonitrile solvent, and is cultured in a 35±2℃ water bath for 72h±1h for simulation extraction to obtain the test solution of the multi-component leachable substance of the intraocular lens.

[0057] S5, Sample test:

[0058] The test solution obtained in S4 is directly injected into the liquid chromatograph at 10ul, and is detected according to the above instrument conditions. Through comparison with the retention time of the control, the leachable substance corresponding to each peak is obtained, and then the content of each component leachable substance is calculated according to the fitting curve (i.e. the standard curve) of each leachable substance control, and the results are shown in Table 3. Figure 3 The test results show that the leachable substances in the to-be-tested sample are HEMA, EOEMA and EGDMA, and the contents thereof are 25.1ug / ml, 40.0ug / ml and 2.7ug / ml respectively, the standard addition recoveries are 103.5%, 102.5% and 98.7% respectively, and the relative standard deviations are 1.5%, 2.2% and 1.8% respectively. It can be known that the method has high detection efficiency, good repeatability and high accuracy.

[0059] The above is only a preferred embodiment of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for determining the content of multi-component leachable matter in an intraocular lens using liquid chromatography, characterized in that, include: Step 1: Weigh out the reference standards for intraocular lens leachables, and then prepare a series of standard solutions of different concentrations for each reference standard using organic solvents; wherein, the leachables of intraocular lenses are 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, glyceryl methacrylate and lauryl methacrylate; Step 2: Gradient elution of the series of standard solutions of each leachable reference standard prepared in Step 1 using liquid chromatography to obtain chromatograms of the leachable substances corresponding to different retention times, and plotting standard curves for each standard solution based on concentration-peak area; wherein the test conditions for liquid chromatography are: The chromatographic column was a CAPCELL PAK ADME, with dimensions of 4.6 mm * 250 mm * 5 µm; mobile phase A was water, and mobile phase B was acetonitrile; the column temperature was 20–50 °C; the flow rate was 0.7–1.5 mL / min; the detector was a DAD detector or a UV detector; the detection wavelength was 210 nm; the injection volume was 1–20 μL; the gradient elution program was as follows: for the first 0–1 min, the volume fraction of mobile phase B was 5%; for the first 1–11 min, the volume fraction of mobile phase B increased from 5% to 100% and held for 6 min; for the last 17–18 min, the volume fraction of mobile phase B decreased from 100% to 5%. Step 3: Add the intraocular lens sample to be tested to an organic solvent and place it in a water bath for simulated extraction to obtain a test solution of the multi-component leachable material of the intraocular lens sample; wherein, the simulated extraction method is as follows: add 1g of the intraocular lens sample to be tested to 10~20ml of organic solvent, place it in a water bath at 35±2℃ for 72h±1h for simulated extraction to obtain a test solution of the multi-component leachable material of the intraocular lens sample; Step 4: Perform liquid chromatography on the test solution obtained in Step 3 to obtain a sample chromatogram; based on the sample chromatogram and the results obtained in Step 2, use the external standard method to qualitatively and quantitatively identify the leachable components in the test solution.

2. The method as described in claim 1, characterized in that, In step 1, the method for preparing the multi-component mixed standard solution is as follows: A mixed standard solution containing the target analyte reference standard is prepared using an organic solvent. The mixed standard solution is then serially diluted with the organic solvent to obtain a series of standard solutions with concentrations ranging from 1 to 50 μg / mL.

3. The method as described in claim 1, characterized in that, In step 1, the leachable reference standard is 2-hydroxyethyl methacrylate reference standard, 2-ethoxyethyl methacrylate reference standard, ethylene glycol dimethacrylate reference standard, trimethylolpropane trimethacrylate reference standard, glyceryl methacrylate reference standard, and lauryl methacrylate reference standard; the organic solvent is one or more selected from methanol, ethanol, isopropanol, acetonitrile, acetone, n-hexane, and dichloromethane.

4. The method according to claim 1, characterized in that, In step 4, the method for qualitative and quantitative analysis of the leachable components in the test solution using the external standard method is as follows: the retention time of each peak in the chromatogram of the sample is compared with the chromatogram of the leachable components in step 2 to determine the type of leachable component corresponding to each peak; then, the content of leachable components in the test solution is calculated based on the peak area and the standard curve in step 2.

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