CN111060614A - Method for analyzing and detecting extract in artificial lens - Google Patents
Method for analyzing and detecting extract in artificial lens Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention relates to the technical field of chemical detection and analysis, in particular to an analysis and detection method for leachates in intraocular lenses. A method for the analytical detection of extractum from an intraocular lens comprising the steps of: (1) artificial lens treatment: extracting the artificial lens by using an extraction solvent to obtain a solution to be detected; (2) solution preparation: preparing a standard stock solution; (3) and (3) detection: and (3) detecting and analyzing the solutions prepared in the step (1) and the step (2) by adopting liquid chromatography and mass spectrometry. The method for analyzing and detecting the extract in the artificial lens has the advantages of high precision, high accuracy, good reproducibility, good stability and high sensitivity.
Description
Technical Field
The invention relates to the technical field of chemical detection and analysis, in particular to an analysis and detection method for leachates in intraocular lenses.
Background
The artificial lens is a special lens made of artificial synthetic materials, has the shape function similar to the lens of human eyes, and has the characteristics of light weight, high optical performance, no antigenicity, inflammation, carcinogenicity, biodegradability and the like. After cataract operation, the turbid crystalline lens is removed, and the artificial crystalline lens is implanted into the eye to replace the original crystalline lens, so that the external objects are focused and imaged on the retina, and the surrounding scenery can be seen clearly. The components of the intraocular lens comprise silica gel, polymethyl methacrylate, hydrogel and the like, and the chemical composition, molecular structure, surface property, processing technology, physicochemical property and the like of the materials can influence the biological safety of the final product.
The biological safety evaluation is to predict the potential hazard of the product in the contact use process with the human body according to the current scientific and technical capability and cognitive level, and simultaneously reduce the unsafe risk to the minimum degree. The evaluation of the biological safety of the medical apparatus is a comprehensive analysis evaluation, and the research of leachables is an indispensable evaluation link. Therefore, the biological safety of the artificial lens can be effectively evaluated by directly evaluating the biological safety of extracts in the artificial lens.
However, no analytical detection method for the extracts in intraocular lenses has been reported in the literature. Therefore, the invention aims to provide the method for analyzing and detecting the extract in the intraocular lens, and the method has the advantages of high precision, high accuracy, good reproducibility, good stability, high sensitivity and the like.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for analyzing and detecting extracts in an intraocular lens, comprising the steps of:
(1) artificial lens treatment: extracting the artificial lens by using an extraction solvent to obtain a solution to be detected;
(2) solution preparation: preparing a standard stock solution;
(3) and (3) detection: and (3) detecting and analyzing the solutions prepared in the step (1) and the step (2) by adopting liquid chromatography and mass spectrometry.
As a preferable technical scheme, in the step (1), the extraction solvent is selected from one or more of ethyl acetate, dichloromethane, ethanol and n-hexane.
As a preferred technical solution, in the extraction solvent, the volume ratio of ethyl acetate to dichloromethane is 1: (3-5).
As a preferred technical scheme, in the step (1), the extraction temperature is 105-120 ℃.
In a preferable embodiment, in step (3), the mobile phase a of the liquid chromatography is an aqueous solution of formic acid.
As a preferable technical scheme, in the step (3), the mobile phase B phase of the liquid chromatogram is a mixture of acetonitrile and methanol.
As a preferable technical solution, in the step (3), the gradient elution conditions of the liquid chromatography are as follows:
0-1 min: mobile phase a 95% → 95%, mobile phase B5% → 5%;
1-6 min: mobile phase a 95% → 60%, mobile phase B5% → 40%;
6-15 min: mobile phase a 60% → 5%, mobile phase B40% → 95%;
15-20 min: mobile phase a was 5% → 5%, and mobile phase B was 95% → 95%.
As a preferred technical solution, in the step (3), the electrospray ion source of the mass spectrum is in a negative ion working mode, and the parameters are as follows: curtain gas: 35.0. mu.L/min, ion source gas 1: 65.0. mu.L/min, ion source gas 2: 60.0. mu.L/min.
As a preferred technical solution, the electrospray ion source parameters of the mass spectrum further include: inlet voltage-10.0V, collision cell outlet voltage: -8.0V.
As a preferred technical scheme, the mass spectrum analysis adopts an MRM mode.
Has the advantages that: according to the method for analyzing and detecting the extract in the intraocular lens, the intraocular lens is extracted by adopting a proper organic solvent, so that the detection accuracy is improved; the accuracy and the sensitivity of detection are improved by elaborately setting liquid chromatography conditions, mass spectrum parameters and the like; meanwhile, due to the mutual synergy among the selection of the extraction solvent, the setting of the liquid chromatography condition and the setting of the mass spectrum parameters, the method for analyzing and detecting the extract in the intraocular lens provided by the invention has the advantages of high precision, high accuracy, good reproducibility, good stability and high sensitivity.
Detailed Description
The technical features of the technical solutions provided by the present invention are further clearly and completely described below with reference to the specific embodiments, and the scope of protection is not limited thereto. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The words "preferred", "more preferred", and the like, in the present invention refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
The invention provides a method for analyzing and detecting extractum in an intraocular lens, which comprises the following steps:
(1) artificial lens treatment: extracting the artificial lens by using an extraction solvent to obtain a solution to be detected;
(2) solution preparation: preparing a standard stock solution;
(3) and (3) detection: and (3) detecting and analyzing the solutions prepared in the step (1) and the step (2) by adopting liquid chromatography and mass spectrometry.
In one embodiment, the intraocular lens extract is 2- [3- (2H-benzotriazol-2-yl) -4-hydroxyphenyl ] ethyl-2-methacrylate.
The CAS number of the 2- [3- (2H-benzotriazole-2-yl) -4-hydroxyphenyl ] ethyl-2-methacrylate is 96478-09-0.
Intraocular lens treatment
The artificial lens treatment refers to extraction treatment of the artificial lens by using an extraction solvent.
In one embodiment, the extraction solvent is selected from one or more of ethyl acetate, dichloromethane, ethanol, n-hexane.
In a preferred embodiment, the extraction solvent is a mixed solvent of ethyl acetate and dichloromethane.
In one embodiment, the volume ratio of ethyl acetate to dichloromethane in the extraction solvent is 1: (3-5).
In a preferred embodiment, the volume ratio of ethyl acetate to dichloromethane in the extraction solvent is 1: 4.
in one embodiment, the extraction temperature is 105-120 ℃.
In a preferred embodiment, the extraction temperature is 110 ℃.
In one embodiment, the volume of the extraction solvent is 160-180 mL.
In a preferred embodiment, the volume of the extraction solvent is 170 mL.
In one embodiment, the intraocular lens has a mass of 200 mg.
In one embodiment, the extraction rate is 4-6 times per hour, with 3-5 hours of extraction.
In a preferred embodiment, the extraction rate is 5 times per hour for 4 hours.
The present invention is not particularly limited to the intraocular lens described.
In one embodiment, the intraocular lens is purchased from major (shanghai) medical devices, inc.
The method adopts common organic solvents such as methanol, acetonitrile, n-hexane and ethyl acetate to extract the intraocular lens, and finds that when the extraction solvent is a mixed solvent of ethyl acetate and dichloromethane, the obtained extracting solution has high transparency, and is favorable for subsequent detection. In particular, the volume ratio of ethyl acetate to dichloromethane is 1: (3-5), the temperature is 105-120 ℃, the accuracy of detection is improved, presumably because: under the temperature, on one hand, the ethyl acetate and the dichloromethane interact to promote the precipitation of substances containing hydroxyl, ester groups and the like, and meanwhile, the system formed by the ethyl acetate and the dichloromethane and the intraocular lens weakens the dissolution driving force of the substances containing carboxyl groups, ethers and the like, so that the extracting solution is transparent, the subsequent detection cannot be blocked, the recovery rate cannot be influenced by extracting redundant impurities, the matrix effect is weakened, and the accuracy is improved. When the ratio of the two is too large, the extracting solution is polluted, and the subsequent detection is not facilitated; when the ratio of the two is too small, the recovery rate is affected because of the large amount of dissolved impurities.
Solution preparation
Standard stock solutions
In a preferred embodiment, the standard stock solution is formulated by the steps of: taking a 2- [3- (2H-benzotriazole-2-yl) -4-hydroxyphenyl ] ethyl-2-methacrylate standard substance into a 10ml volumetric flask, dissolving the standard substance by using a mixed solvent of n-hexane and isopropanol, fixing the volume, shaking up, sealing, keeping out of the sun, and placing the standard substance into a refrigerator for storage to prepare 5 series of standard stock solutions with concentration gradients, wherein the standard stock solutions are marked as linear 1, linear 2, linear 3, linear 4 and linear 5 respectively, and the specific table is shown in table 1.
In one embodiment, the volume ratio of n-hexane to isopropanol is 1: 1.
TABLE 1 concentration of extract in Standard stock solution (. mu.g/mL)
| Linear 1 | Linearity 2 | Line 3 | Linearity 4 | Linear 5 |
| 2855 | 2370 | 2228 | 1670 | 100 |
In one embodiment, the 2- [3- (2H-benzotriazol-2-yl) -4-hydroxyphenyl ] ethyl-2-methacrylate standard has a CAS number of 96478-09-0, available from Sigma-Aldrich.
The standard solution stock solution refers to a reagent solution with an accurately known concentration, and is prepared for the purpose of facilitating quantitative analysis, and a working curve is drawn or a calculation standard is made using the standard solution so as to perform quantitative analysis on a target compound.
In a preferred embodiment, the preparing of the solution further comprises preparing a sample solution, preparing a system applicability solution, preparing a detection limit solution, preparing a quantification limit solution, and preparing an accuracy solution.
Sample solution
In a preferred embodiment, the preparation of the sample solution comprises the steps of: and (3) concentrating 100ml of solution to be detected to 5ml by using a rotary evaporator, and then metering the volume to 25ml by using a solvent to obtain a sample solution.
In one embodiment, the solvent is a mixed solvent of n-hexane and isopropanol.
In one embodiment, the volume ratio of n-hexane to isopropanol is 1: 1.
system applicability solution
In a preferred embodiment, the formulation of the system suitability solution comprises the steps of: precisely transferring 200 μ L of standard stock solution linear 5 into a 20ml volumetric flask, shaking up with mixed solvent of n-hexane and isopropanol to constant volume, then precisely transferring 5ml of the solution into a 50ml volumetric flask, and shaking up with mixed solvent of n-hexane and isopropanol to constant volume.
In one embodiment, the volume ratio of n-hexane to isopropanol is 1: 1.
detection limit solution
In a preferred embodiment, the preparation of the detection limit solution comprises the steps of: precisely transferring 2ml of the system applicability solution into a 10ml volumetric flask, metering the volume with a solvent, and shaking up to obtain the detection limit solution.
In one embodiment, the solvent is a mixed solvent of n-hexane and isopropanol.
In one embodiment, the volume ratio of n-hexane to isopropanol is 1: 1.
quantitative limiting solution
In a preferred embodiment, the preparation of the quantitative limiting solution comprises the following steps: concentrating 100ml of solution to be detected to 5ml by using a rotary evaporator, adding 4ml of n-hexane system applicability solution, metering the volume to 25ml by using a solvent, and preparing three times in parallel by the same method.
In one embodiment, the solvent is a mixed solvent of n-hexane and isopropanol.
In one embodiment, the volume ratio of n-hexane to isopropanol is 1: 1.
accuracy solution
In a preferred embodiment, the preparation of the accuracy solution comprises the steps of: concentrating 100ml of solution to be detected to 5ml by using a rotary evaporator, adding 12ml of n-hexane system applicability solution, metering the volume to 25ml by using a solvent, and preparing six times in parallel by the same method.
In one embodiment, the solvent is a mixed solvent of n-hexane and isopropanol.
In one embodiment, the volume ratio of n-hexane to isopropanol is 1: 1.
detection of
< liquid chromatography conditions >
In a preferred embodiment, the liquid chromatography instrument Agilent 1290 and 6545B ultra performance liquid chromatography is adopted in the invention.
In a preferred embodiment, the chromatographic column of the liquid chromatograph is Poroshell120, C18, 2.7 μm, 3.0 × 150 mm.
In a preferred embodiment, the flow rate is 0.4 ml/min.
In a preferred embodiment, the column temperature is 40 ℃.
In a preferred embodiment, the sample introduction volume is 5 μ l.
In a preferred embodiment, the mobile phase a of the liquid chromatography is aqueous formic acid.
In a preferred embodiment, the concentration of the aqueous formic acid solution is from 4 to 6 mmol/L.
In a more preferred embodiment, the concentration of the aqueous formic acid solution is 5 mmol/L.
In a preferred embodiment, the mobile phase B of the liquid chromatography is a mixture of acetonitrile and methanol.
In a preferred embodiment, the volume ratio of acetonitrile to methanol is (6-8): (2-4).
In a more preferred embodiment, the volume ratio of acetonitrile to methanol is 7: 3.
in a preferred embodiment, the gradient elution conditions of the liquid chromatography are:
0-1 min: mobile phase a 95% → 95%, mobile phase B5% → 5%;
1-6 min: mobile phase a 95% → 60%, mobile phase B5% → 40%;
6-15 min: mobile phase a 60% → 5%, mobile phase B40% → 95%;
15-20 min: mobile phase a was 5% → 5%, and mobile phase B was 95% → 95%.
The retention time is prolonged by the combination of the hydroxyl group of 2- [3- (2H-benzotriazol-2-yl) -4-hydroxyphenyl ] ethyl-2-methacrylate and the polar group in the organic phase, and the phenomenon of too fast elution is balanced by the addition of acetonitrile with a stabilizing group. The applicant finds that the accuracy and the sensitivity of detection can be improved by selecting a 5mmol/L formic acid aqueous solution and matching with a certain gradient elution condition. The guess is that: through fixed mobile A looks and B looks to and certain elution condition, begin with 95% A looks at, end with 95% B looks at for substance and mobile phase to be measured are when the chromatographic column, the activity of inhibition silicon hydroxyl that can be fine reduces the tailing, improves the peak shape, has improved the SNR for reduce, can wash the impurity of minipolarity in addition, prevents that the sample from remaining to next needle.
Although the mixed solvent of ethyl acetate and dichloromethane can effectively extract 2- [3- (2H-benzotriazole-2-yl) -4-hydroxyphenyl ] ethyl-2-methacrylate, other impurities in the intraocular lens can hardly be separated out, the substances and the analyte flow out of the spray needle together to compete with charges, atomization and volatilization of the analyte are influenced, ions finally entering a mass spectrum are reduced or enhanced, and thus a matrix inhibition or enhancement effect is generated.
In a preferred embodiment, the DAD detection wavelength is 190-400 nm.
< Mass Spectrometry Condition >
In a preferred embodiment, the electrospray ion source of the mass spectrometer has a negative ion working mode, and the parameters are as follows: curtain gas: 35.0. mu.L/min, ion source gas 1: 65.0. mu.L/min, ion source gas 2: 60.0. mu.L/min.
In a preferred embodiment, the electrospray ion source parameters of mass spectrometry further comprise: inlet voltage-10.0V, collision cell outlet voltage: -8.0V.
In a preferred embodiment, the analysis of the mass spectrum uses the MRM mode.
In a preferred embodiment, the scanning mode of the mass spectrum is full scan, and the scanning range is 50-1500 m/z.
The applicant can further improve the accuracy of the detection method by elaborately setting the mass spectrum parameters, probably because the parameters of the ion source gas 1, the ion source gas 2, the curtain gas and the like are mutually cooperated, on one hand, the atomization of the solvent is accelerated and high-charge liquid drops are generated, on the other hand, the entrance of neutral molecules, small liquid drops, solid particles and the like is prevented, and the stability of signals is improved; generally, when monitoring the MRM reaction, the entrance voltage and the exit voltage of the collision cell are set to be low, so that the reaction is sufficient, but this results in ion loss; the inlet voltage is set to be-10.0V under the coordination of other parameters; when the outlet voltage of the collision chamber is-8.0V, ions to be detected can fully collide and react without increasing ion loss during collision, and finally, a secondary spectrogram with abundant fragments and characteristic fragment ions are obtained, so that characteristic peak matching is facilitated, and the detection sensitivity is improved.
If the inlet and outlet voltages are too high, the ions cannot collide sufficiently; if the entrance and exit voltages are too low, ion loss may result from excessive collisions.
System suitability detection
In a preferred embodiment, the system suitability detection method is as follows: the prepared system suitability test solution was measured under the above measurement conditions, and the response value and RSD calculation result were calculated and reported.
Detection limit detection method
In a preferred embodiment, the detection limit detection method is as follows: and (3) measuring the detection limit solution prepared according to the measuring conditions, wherein the concentration which is more than 3 times of the signal to noise ratio is used as the detection limit.
Quantitative limit detection method
In a preferred embodiment, the quantitative limit detection method is as follows: according to the above-mentioned measuring conditions, the quantitative limiting solution obtained by preparation is measured, and the concentration greater than 10 times of signal-to-noise ratio is used as quantitative limit.
Accuracy detection method
In a preferred embodiment, the accuracy detection method is as follows: and (4) sampling solution and accuracy solution, analyzing sample introduction, and calculating the recovery rate and the RSD.
Linear detection method
In a preferred embodiment, the linear detection method is as follows: and (3) sampling and analyzing the standard stock solution, and automatically calculating a regression equation by using an instrument by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, wherein the correlation coefficient (r) of the equation is not lower than 0.99.
Hereinafter, the present invention will be described in more detail by way of examples, but it should be understood that these examples are merely illustrative and not restrictive. In addition, the starting materials used are all commercially available, unless otherwise specified.
Examples
Example 1
Embodiment 1 of the present invention provides a method for analyzing and detecting extractum in an intraocular lens, comprising the following steps:
(1) artificial lens treatment: extracting the artificial lens by using an extraction solvent to obtain a solution to be detected;
(2) solution preparation: preparing a standard stock solution;
(3) and (3) detection: and (3) detecting and analyzing the solutions prepared in the step (1) and the step (2) by adopting liquid chromatography and mass spectrometry.
The extract in the artificial lens is 2- [3- (2H-benzotriazole-2-yl) -4-hydroxyphenyl ] ethyl-2-methacrylate.
In the step (1), the extraction solvent is a mixed solvent of ethyl acetate and dichloromethane.
The volume ratio of the ethyl acetate to the dichloromethane is 1: 4.
the extraction temperature was 110 ℃.
The volume of the extraction solvent was 170 mL.
The mass of the artificial lens is 200 mg.
The extraction rate was 5 times per hour, with 4 hours of extraction.
In the step (2), the preparation of the standard stock solution comprises the following steps: taking a 2- [3- (2H-benzotriazole-2-yl) -4-hydroxyphenyl ] ethyl-2-methacrylate standard substance into a 10ml volumetric flask, dissolving the standard substance by using a mixed solvent of n-hexane and isopropanol, fixing the volume, shaking up, sealing, keeping out of the sun, and placing the standard substance into a refrigerator for storage to prepare 5 series of standard stock solutions with concentration gradients, wherein the standard stock solutions are marked as linear 1, linear 2, linear 3, linear 4 and linear 5 respectively, and the specific table is shown in table 1.
The volume ratio of the n-hexane to the isopropanol is 1: 1.
TABLE 1 concentration of extract in Standard stock solution (. mu.g/mL)
| Linear 1 | Linearity 2 | Line 3 | Linearity 4 | Linear 5 |
| 2855 | 2370 | 2228 | 1670 | 100 |
In the step (2), the preparation of the sample solution comprises the following steps: and (3) concentrating 100ml of solution to be detected to 5ml by using a rotary evaporator, and then metering the volume to 25ml by using a solvent to obtain a sample solution.
The solvent is a mixed solvent of n-hexane and isopropanol.
The volume ratio of the n-hexane to the isopropanol is 1: 1.
in the step (2), the preparation of the system suitability solution comprises the following steps: precisely transferring 200 μ L of standard stock solution linear 5 into a 20ml volumetric flask, shaking up with mixed solvent of n-hexane and isopropanol to constant volume, then precisely transferring 5ml of the solution into a 50ml volumetric flask, and shaking up with mixed solvent of n-hexane and isopropanol to constant volume.
The volume ratio of the n-hexane to the isopropanol is 1: 1.
in the step (2), the preparation of the detection limit solution comprises the following steps: precisely transferring 2ml of the system applicability solution into a 10ml volumetric flask, metering the volume with a solvent, and shaking up to obtain the detection limit solution.
The solvent is a mixed solvent of n-hexane and isopropanol.
The volume ratio of the n-hexane to the isopropanol is 1: 1.
in the step (2), the preparation of the quantitative limiting solution comprises the following steps: concentrating 100ml of solution to be detected to 5ml by using a rotary evaporator, adding 4ml of n-hexane system applicability solution, metering the volume to 25ml by using a solvent, and preparing three times in parallel by the same method.
The solvent is a mixed solvent of n-hexane and isopropanol.
The volume ratio of the n-hexane to the isopropanol is 1: 1.
in the step (2), the preparation of the accuracy solution comprises the following steps: concentrating 100ml of solution to be detected to 5ml by using a rotary evaporator, adding 12ml of n-hexane system applicability solution, metering the volume to 25ml by using a solvent, and preparing six times in parallel by the same method.
The solvent is a mixed solvent of n-hexane and isopropanol.
The volume ratio of the n-hexane to the isopropanol is 1: 1.
in the step (3), the conditions of the liquid chromatography are as follows:
a) the liquid chromatography instrument is an Agilent 1290 and 6545B ultra-high performance liquid chromatography instrument.
b) The column was Poroshell120, C18, 2.7 μm, 3.0X 150 mm.
c) The flow rate was 0.4 ml/min.
d) The column temperature was 40 ℃.
e) The injection volume was 5. mu.l.
f) The mobile phase A is 5mmol/L formic acid aqueous solution.
g) The mobile phase B is a mixture of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol is 7: 3.
h) the gradient elution conditions were:
0-1 min: mobile phase a 95% → 95%, mobile phase B5% → 5%;
1-6 min: mobile phase a 95% → 60%, mobile phase B5% → 40%;
6-15 min: mobile phase a 60% → 5%, mobile phase B40% → 95%;
15-20 min: mobile phase a was 5% → 5%, and mobile phase B was 95% → 95%.
In the step (3), the DAD detection wavelength is 190-400 nm.
In the step (3), the mass spectrum conditions are as follows: the electrospray ion source of the mass spectrum is in a negative ion working mode, and the parameters are as follows: curtain gas: 35.0. mu.L/min, ion source gas 1: 65.0. mu.L/min, ion source gas 2: 60.0. mu.L/min, inlet voltage: -10.0V, collision cell exit voltage: 8.0V, MRM mode is adopted, the scanning mode is full scanning, and the scanning range is 50-1500 m/z.
Example 2
Embodiment 2 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is the same as embodiment 1 in specific embodiment, except that in step (1), the volume ratio of ethyl acetate to dichloromethane is 1: 1.
example 3
Embodiment 3 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is the same as embodiment 1 in the specific implementation manner, except that in step (1), the volume ratio of ethyl acetate to dichloromethane is 1: 8.
example 4
Embodiment 4 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is similar to embodiment 1, except that the extraction solvent in step (1) is ethyl acetate.
Example 5
Example 5 of the present invention provides a method for analyzing and detecting extracts from intraocular lenses, which is similar to example 1, except that in step (1), the extraction solvent is dichloromethane.
Example 6
Embodiment 6 of the present invention provides an analysis and detection method for extracts in intraocular lenses, which is the same as embodiment 1 in specific implementation, except that in step (1), the extraction solvent is methanol and n-hexane, and the volume ratio of methanol to n-hexane is 1: 4.
example 7
Embodiment 7 of the present invention provides an analysis and detection method for extracts in intraocular lenses, which is the same as embodiment 1 in specific implementation, except that in step (1), the extraction solvent is n-hexane and ethyl acetate, and the volume ratio of n-hexane to ethyl acetate is 1: 4.
example 8
Embodiment 8 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is the same as embodiment 1 in specific implementation manner, except that in step (1), the extraction solvent is acetonitrile and dichloromethane, and the volume ratio of acetonitrile to dichloromethane is 1: 4.
example 9
Example 9 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is similar to example 1 except that in step (3), the mobile phase a is 2mmol/L formic acid aqueous solution.
Example 10
Example 10 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is similar to example 1, except that in step (3), the mobile phase A is 8mmol/L formic acid aqueous solution.
Example 11
Example 11 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is similar to example 1, except that in step (3), the gradient elution conditions are as follows:
0-1 min: mobile phase a 5% → 5%, mobile phase B95% → 95%;
1-6 min: mobile phase a 5% → 60%, mobile phase B95% → 40%;
6-15 min: mobile phase a 60% → 95%, mobile phase B40% → 5%;
15-20 min: mobile phase a was 95% → 95%, and mobile phase B was 5% → 5%.
Example 12
Embodiment 12 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is the same as embodiment 1, except that in step (3), the mobile phase B is a mixture of acetonitrile and methanol, and the volume ratio of acetonitrile to methanol is 5: 5.
example 13
Embodiment 13 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is the same as embodiment 1, except that in step (3), the mobile phase B is a mixture of acetonitrile and methanol, and the volume ratio of acetonitrile to methanol is 9: 1.
example 14
Embodiment 14 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is similar to embodiment 1, except that in step (3), the column temperature is 30 ℃ and the sample injection volume is 3 μ l.
Example 15
Embodiment 15 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is the same as embodiment 1 except that in step (3), the column temperature is 50 ℃ and the sample injection volume is 8 μ l.
Example 16
Example 16 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is similar to example 1, except that in step (3), the inlet voltage is-10.0V and the outlet voltage of the collision cell is-3.0V.
Example 17
Example 17 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is similar to example 1, except that in step (3), the inlet voltage is-15.0V and the outlet voltage of the collision cell is-12.0V.
Example 18
Embodiment 18 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is implemented in the same manner as in embodiment 1, except that, in step (3), the air curtain: 30.0. mu.L/min, ion source gas 1: 55.0. mu.L/min, ion source gas 2: 55.0. mu.L/min.
Example 19
Embodiment 19 of the present invention provides a method for analyzing and detecting extracts in intraocular lenses, which is similar to embodiment 1, except that in step (3), the air curtain: 40.0. mu.L/min, ion source gas 1: 75.0 μ L/min, ion source gas 2: 75.0. mu.L/min.
Performance evaluation
1. System suitability detection
The formulated system suitability test solution was tested, and response values and RSD calculations were calculated and reported according to the method for analytical detection of extractants from intraocular lenses described in example 1. The results are shown in Table 2.
Table 2 results of system applicability test of example 1
2. Detection limit detection
The detection limit solutions prepared were measured according to the methods for analyzing and detecting extracts in intraocular lenses described in examples 1 and 9-15, respectively, with the concentration greater than 3 times the signal-to-noise ratio being the detection limit. The results are shown in Table 3.
TABLE 3 detection Limit test results for examples 1, 9-15
3. Quantitative limit detection
The quantitative limit solutions prepared were measured according to the analytical detection methods for intraocular lens extracts described in examples 1, 9-15, respectively, with concentrations greater than 10 times the signal-to-noise ratio being the quantitative limit, wherein the recovery measurements were averaged three times. The results are shown in Table 4.
TABLE 4 results of detection of limit of quantitation in examples 1, 9 to 15
| Recovery (%) | RSD(％) | Quantitative concentration (μ g/ml) | |
| Example 1 | 97 | 2 | 0.06 |
| Example 9 | 92 | 4 | 0.09 |
| Example 10 | 114 | 7 | 0.15 |
| Example 11 | 83 | 8 | 0.18 |
| Example 12 | 113 | 6 | 0.12 |
| Example 13 | 93 | 5 | 0.08 |
| Example 14 | 90 | 5 | 0.10 |
| Example 15 | 91 | 4 | 0.09 |
4. Accuracy detection
The sample solutions and the accuracy solutions obtained by the preparation were subjected to sample injection analysis and the recovery rates and RSD were calculated according to the analysis and detection methods for extracts in intraocular lenses described in examples 1 to 15, respectively, wherein the recovery rate measurement was taken as an average value for six times. The results are shown in Table 5.
TABLE 5 results of accuracy tests of examples 1-15
5. Linear detection
According to the method for analyzing and detecting the extract in the intraocular lens described in example 1, a standard stock solution is taken for sample injection analysis, the concentration is taken as a horizontal coordinate, the peak area is taken as a vertical coordinate, an instrument automatically calculates a regression equation, and the correlation coefficient (r) of the equation is not lower than 0.99. The results are shown in Table 6.
Table 6 results of linear detection of example 1
| Linear equation of equations | Correlation coefficient |
| y＝331.0888x+323.1074 | R＝0.9997 |
The method for analyzing and detecting the extractum in the intraocular lens in the embodiment 1 can obtain the characteristic fragment ion peak with better signal intensity, has lower noise and more accurate baseline; the methods for analyzing and detecting extracts in intraocular lenses described in examples 16-19 had relatively high noise and shifted baseline.
As can be seen from tables 2-6, the method for analyzing and detecting the extract in the intraocular lens provided by the invention has the advantages that the minimum detected concentration of the extract is 0.02 mu g/ml, the minimum quantitative concentration is 0.06 mu g/ml, the recovery rate is 102%, the standard relative deviation is not more than 5%, and the correlation coefficient is more than 0.99. The method has the advantages of high precision, high accuracy, good reproducibility, good stability and high sensitivity.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.
Claims (10)
1. A method for analyzing and detecting extracts in an intraocular lens, comprising the steps of:
(1) artificial lens treatment: extracting the artificial lens by using an extraction solvent to obtain a solution to be detected;
(2) solution preparation: preparing a standard stock solution;
(3) and (3) detection: and (3) detecting and analyzing the solutions prepared in the step (1) and the step (2) by adopting liquid chromatography and mass spectrometry.
2. The method for analyzing and detecting intraocular lens extract according to claim 1 wherein in step (1) the extraction solvent is selected from one or more of ethyl acetate, dichloromethane, ethanol, n-hexane.
3. The method for analyzing and detecting intraocular lens extract according to claim 2 wherein the volume ratio of ethyl acetate to methylene chloride in the extraction solvent is 1: (3-5).
4. The method for analyzing and detecting extractants from intraocular lens according to claim 1 or 2, wherein in step (1), the extraction temperature is 105-120 ℃.
5. The method for analyzing and detecting intraocular lens extract according to claim 1 wherein in step (3), the mobile phase a of the liquid chromatography is aqueous formic acid.
6. The method for analyzing and detecting intraocular lens extract according to claim 1 wherein in step (3) the mobile phase B of the liquid chromatography is a mixture of acetonitrile and methanol.
7. The method for analyzing and detecting intraocular lens extract according to claim 1 wherein in step (3) the gradient elution conditions of the liquid chromatogram are:
0-1 min: mobile phase a 95% → 95%, mobile phase B5% → 5%;
1-6 min: mobile phase a 95% → 60%, mobile phase B5% → 40%;
6-15 min: mobile phase a 60% → 5%, mobile phase B40% → 95%;
15-20 min: mobile phase a was 5% → 5%, and mobile phase B was 95% → 95%.
8. The method for analyzing and detecting extractants in intraocular lens according to claim 1, wherein in step (3), the electrospray ion source of mass spectrometry is in negative ion working mode, and the parameters are as follows: curtain gas: 35.0. mu.L/min, ion source gas 1: 65.0. mu.L/min, ion source gas 2: 60.0. mu.L/min.
9. The method of claim 8, wherein the mass spectrometric electrospray ion source parameters further comprise: inlet voltage-10.0V, collision cell outlet voltage: -8.0V.
10. The method of claim 1, wherein the mass spectrometric analysis uses MRM mode.
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