CN115684400A - HPLC detection method for ketoprofen ethyl ester in water and algae culture medium - Google Patents
HPLC detection method for ketoprofen ethyl ester in water and algae culture medium Download PDFInfo
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Abstract
The invention discloses an HPLC detection method of ketoprofen ethyl ester in water and algae culture medium, which uses a ZORBAX SB-C18 chromatographic column, takes water and methanol as mobile phases, the flow rate is 0.9-1.2 mL/min, the temperature of the chromatographic column is 30-40 ℃, and the detection wavelength is 254nm. The detection method has good linear relation within the range of 0.100 mg/L-2.00 mg/L, the linear equation is y =110299x +511.061, and the correlation coefficient R =0.9999; according to the method, the ketoprofen ethyl ester peaks at about 7.45min, and a blank sample has no interference peak at the time point, so that the method specificity is strong; LOD is 3.97 mu g/L; LOQ is 13.2 mu g/L; the method has the advantages that the average adding recovery rate of the ketoprofen ethyl ester in the aerated water is 95.4-97.1%, the average adding recovery rate in the algae culture medium is 88.6-94.4%, the method realizes effective detection of the ketoprofen ethyl ester, the detection result is good in accuracy, high in sensitivity, simple and convenient to operate, low in cost, short in analysis time and good in repeatability, and guarantees the quality control of the ketoprofen ethyl ester.
Description
Technical Field
The invention belongs to the field of organic compound analysis, and particularly relates to an HPLC (high performance liquid chromatography) detection method for ketoprofen ethyl ester in water and an algae culture medium.
Background
Ketoprofen ethyl ester, chemical name of which is 3-benzoyl-alpha-methyl phenylacetic acid ethyl ester, molecular formula of which is C 18 H 18 O 3 It is often used as an intermediate of ketoprofen to synthesize ketoprofen and its chiral enantiomers. The structural formula is as follows:
ketoprofen is a typical nonsteroidal anti-inflammatory, antipyretic and analgesic drug, and is widely applied to the treatment of diseases such as rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, gout and the like.
Chinese patent ZL201110222136.3 is a method for preparing ketoprofen ethyl ester, and discloses a method for preparing ketoprofen ethyl ester by reacting 2- (3-benzoylphenyl) propionitrile with ethanol in the presence of sulfuric acid, but does not disclose a method for controlling the quality of ketoprofen ethyl ester.
At present, only the conventional physicochemical indexes of the ketoprofen ethyl ester are usually detected, but the quality condition of the ketoprofen ethyl ester is difficult to reflect really only through the conventional physicochemical indexes, so that a method capable of accurately performing qualitative and quantitative detection on the ketoprofen ethyl ester is established, and the method has great guiding significance for the production and application of the ketoprofen ethyl ester.
Disclosure of Invention
The invention aims to make up the defects of the prior art, provides an HPLC detection method of ketoprofen ethyl ester in water and algae culture medium, and more accurately realizes the quality control of the ketoprofen ethyl ester.
The purpose of the invention is achieved by the following technical scheme:
an HPLC detection method of ketoprofen ethyl ester in water and algae culture medium adopts a high performance liquid chromatograph and a PDA detector, and detects the ketoprofen ethyl ester by a ZORBAX SB-C18, 250mm multiplied by 4.6mm and 5 μm chromatographic column, and the HPLC detection adopts the following chromatographic conditions: the mobile phase A is methanol, and the mobile phase B is water; the flow rate is 0.8-1.2 mL/min; the temperature of the chromatographic column is 30-40 ℃; the detection wavelength is 254nm, and the sample injection amount is 25-35 mu L.
The volume ratio of the mobile phase methanol to the water is 75.
Preferably, the mobile phase flow rate is 1.0mL/min.
Preferably, the temperature of the chromatographic column is 32 to 38 ℃.
More preferably, the column temperature is 35 ℃.
Preferably, the sample amount is 30 μ L.
The detection method comprises the following steps: (1) preparation of test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving the sample with methanol, fixing the volume to the scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking an appropriate amount of the sample stock solution, diluting the sample stock solution with a diluent to obtain a sample solution, and waiting for HPLC (high performance liquid chromatography) determination. (2) preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution with methanol into a series of standard working solutions with the concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L, and waiting for HPLC determination. (3) measurement and calculation: setting instrument parameters, after the instrument is stabilized, carrying out HPLC (high performance liquid chromatography) determination on the test sample solution and the standard sample solution, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Preferably, the diluent is aerated water or an algal culture medium.
Under the chromatographic condition of the method, the retention time of the ketoprofen ethyl ester is 7.45min.
The LOD of the ketoprofen ethyl ester by the method is 3.97 mu g/L, and the LOQ is 13.2 mu g/L.
Advantageous effects
1. The invention establishes an HPLC detection method for ketoprofen ethyl ester in water and algae culture medium, overcomes the problem that the quality control of ketoprofen ethyl ester is still blank in the prior art, and provides reference for the quality control in the production application process and related research processes of ketoprofen ethyl ester. Proved by a proof chemical test, the method has the advantages of strong specificity, good repeatability, good linear relation and strong stability, and can meet the quality control requirement of the ketoprofen ethyl ester.
2. Linear tests of the invention show that the linear relation is good in the range of 0.100 mg/L-2.00 mg/L, the linear equation is y =110299x +511.061, and the linear correlation coefficient R =0.9999 of the curve.
3. By comparing spectrograms of a blank sample of aerated water and an algae culture medium and a sample with a recovery rate, the method has the advantages that the ketoprofen ethyl ester peaks at 7.45min, and the blank sample has no interference peak at the time point, so that the method has good specificity on the ketoprofen ethyl ester.
4. The RSDR.T. of the target peak of the method is 0.11%, the RSDArea is 0.80%, and the RSDArea is less than 5.00%, and the result shows that the method can be used for qualitative and quantitative analysis of ketoprofen ethyl ester; the LOD of the method is 3.97 mu g/L; LOQ is 13.2 mug/L, and can meet the detection requirement.
5. The recovery rate test of the invention shows that the average addition recovery rate of the ketoprofen ethyl ester with different concentrations in the aerated water is 95.4-97.1%, and the average addition recovery rate in the algae culture medium is 88.6-94.4%.
6. The stability test of the invention shows that the detection and analysis method of the invention can respectively meet the concentration determination requirements of daphnia acute activity inhibition test, algae growth inhibition test and fish acute toxicity test.
Drawings
Fig. 1 is a linear fit plot of ketoprofen ethyl ester;
FIG. 2 is a typical map of ketoprofen ethyl ester;
FIG. 3 is a typical spectrum of a recovery spiked solution in aerated water of low-concentration ketoprofen ethyl ester;
FIG. 4 is a typical spectrum of a recovery spiked solution in aerated water of high-concentration ketoprofen ethyl ester;
FIG. 5 is a typical spectrum of recovery spiked solution in low concentration ketoprofen ethyl acetate medium;
FIG. 6 is a typical spectrum of recovery spiked solution in low concentration ketoprofen ethyl acetate medium;
FIG. 7 is a stability profile (0 h) of ketoprofen ethofish under test conditions;
FIG. 8 is a stability map of ketoprofen ethyl ester fish under test conditions (96 h);
FIG. 9 is a stability map (0 h) of ketoprofen ethyl daphnia in a test environment;
FIG. 10 is a stability map (48 h) of ketoprofen ethyl daphnia in a test environment;
FIG. 11 is a stability profile (0 h) of ketoprofen ethyl alga under test conditions;
FIG. 12 is a stability profile of ketoprofen ethyl acetate algae under test conditions (72 h).
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. The reagents and instruments used in the examples are not indicated by the manufacturer, and conventional products are commercially available. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Instruments and reagents involved in the following examples include, but are not limited to:
instruments and reagents used in the examples:
1. reagents and solvents
(1) Methanol: HPLC, shanghai' an Spectrum Experimental science & technology, inc.;
(2) UP water: resistivity, 18.2M Ω cm;
(3) Aeration water: self-made in a laboratory;
(4) Algae culture medium: self-made in a laboratory;
(5) Ketoprofen ethyl ester, purity 99.2%.
2. Main instrument equipment
(1) High performance liquid chromatograph: shimadzu LC2030C 3D Plus;
(2) A SHAMD Labsolutions data acquisition, qualitative analysis and quantitative analysis workstation;
(3) An electronic balance: mettler-tollido (china) ltd, XSE205D, UEL;
(4) Biochemical incubator, ZXSD-B1430.
Example 1
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 1.0mL/min; the temperature of the chromatographic column is 35 ℃; the detection wavelength was 254nm and the sample size was 30. Mu.L.
(2) Preparation of a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving the sample with methanol, fixing the volume to the scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking a proper amount of the sample stock solution, diluting the sample stock solution with aerated water to obtain a sample solution, and measuring according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentrations of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC determination;
(4) Measurement and calculation: and setting instrument parameters according to HPLC chromatographic conditions, performing HPLC determination on the test solution and the standard solution after the instrument is stabilized, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 2
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 0.8mL/min; the temperature of the chromatographic column is 30 ℃; the detection wavelength was 254nm, and the sample size was 25. Mu.L.
(2) Preparing a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving the sample with methanol, fixing the volume to the scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking a proper amount of the sample stock solution, diluting the sample stock solution with aerated water to obtain a sample solution, and measuring according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to the scale, shaking up, and preparing ketoprofen ethyl ester standard stock solution with the concentration of 1000 mg/L; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC (high performance liquid chromatography) determination;
(4) Measurement and calculation: setting instrument parameters according to HPLC chromatographic conditions, carrying out HPLC determination on the test solution and the standard solution after the instrument is stable, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 3
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 mu m; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 0.9mL/min; the temperature of the chromatographic column is 32 ℃; the detection wavelength was 254nm and the sample size was 28. Mu.L.
(2) Preparation of a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving the sample with methanol, fixing the volume to the scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking a proper amount of the sample stock solution, diluting the sample stock solution with aerated water to obtain a sample solution, and measuring according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC (high performance liquid chromatography) determination;
(4) Measurement and calculation: setting instrument parameters according to HPLC chromatographic conditions, carrying out HPLC determination on the test solution and the standard solution after the instrument is stable, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 4
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 1.1mL/min; the temperature of the chromatographic column is 38 ℃; the detection wavelength was 254nm and the amount of sample was 32. Mu.L.
(2) Preparation of a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving the sample with methanol, fixing the volume to the scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking a proper amount of the sample stock solution, diluting the sample stock solution with aerated water to obtain a sample solution, and measuring according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC (high performance liquid chromatography) determination;
(4) Measurement and calculation: setting instrument parameters according to HPLC chromatographic conditions, carrying out HPLC determination on the test solution and the standard solution after the instrument is stable, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 5
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 1.2mL/min; the temperature of the chromatographic column is 40 ℃; the detection wavelength was 254nm and the sample size was 35. Mu.L.
(2) Preparation of a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving the sample with methanol, fixing the volume to the scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking a proper amount of the sample stock solution, diluting the sample stock solution with aerated water to obtain a sample solution, and measuring according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC (high performance liquid chromatography) determination;
(4) Measurement and calculation: and setting instrument parameters according to HPLC chromatographic conditions, performing HPLC determination on the test solution and the standard solution after the instrument is stabilized, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 6
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 1.0mL/min; the temperature of the chromatographic column is 35 ℃; the detection wavelength was 254nm, and the sample size was 30. Mu.L.
(2) Preparation of a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking an appropriate amount of the sample stock solution, diluting with an algae culture medium to obtain a test solution, and determining according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to the scale, shaking up, and preparing ketoprofen ethyl ester standard stock solution with the concentration of 1000 mg/L; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentrations of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC determination;
(4) Measurement and calculation: setting instrument parameters according to HPLC chromatographic conditions, carrying out HPLC determination on the test solution and the standard solution after the instrument is stable, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 7
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 0.8mL/min; the temperature of the chromatographic column is 30 ℃; the detection wavelength was 254nm, and the sample size was 25. Mu.L.
(2) Preparing a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking an appropriate amount of the sample stock solution, diluting with an algae culture medium to obtain a test solution, and determining according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC (high performance liquid chromatography) determination;
(4) Measurement and calculation: setting instrument parameters according to HPLC chromatographic conditions, carrying out HPLC determination on the test solution and the standard solution after the instrument is stable, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 8
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 0.9mL/min; the temperature of the chromatographic column is 32 ℃; the detection wavelength was 254nm and the sample size was 28. Mu.L.
(2) Preparation of a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking an appropriate amount of the sample stock solution, diluting with an algae culture medium to obtain a test solution, and determining according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC (high performance liquid chromatography) determination;
(4) Measurement and calculation: setting instrument parameters according to HPLC chromatographic conditions, carrying out HPLC determination on the test solution and the standard solution after the instrument is stable, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 9
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 1.1mL/min; the temperature of the chromatographic column is 38 ℃; the detection wavelength was 254nm, and the sample size was 32. Mu.L.
(2) Preparation of a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking an appropriate amount of the sample stock solution, diluting with an algae culture medium to obtain a test solution, and determining according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC (high performance liquid chromatography) determination;
(4) Measurement and calculation: setting instrument parameters according to HPLC chromatographic conditions, carrying out HPLC determination on the test solution and the standard solution after the instrument is stable, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
Example 10
(1) HPLC chromatographic conditions: the chromatographic column is ZORBAX SB-C18, 250mm is multiplied by 4.6mm, and 5 μm; the mobile phase is A: methanol, B: water; a: B =75 (v/v); the flow rate is 1.2mL/min; the temperature of the chromatographic column is 40 ℃; the detection wavelength was 254nm, and the sample size was 35. Mu.L.
(2) Preparing a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking an appropriate amount of the sample stock solution, diluting with an algae culture medium to obtain a test solution, and determining according to HPLC chromatographic conditions;
(3) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentrations of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC determination;
(4) Measurement and calculation: and setting instrument parameters according to HPLC chromatographic conditions, performing HPLC determination on the test solution and the standard solution after the instrument is stabilized, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
To further prove the feasibility of the invention, the inventors have verified the detection method of the invention by a number of analytical method tests, and part of the test contents are summarized as follows:
1. linear test
Precisely sucking 1.00mL of standard working solution with the series concentration of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L in example 1, performing high performance liquid chromatography measurement according to the detection method of the invention, drawing a calibration curve by taking the concentration of ketoprofen ethyl ester as an abscissa and the peak area of ketoprofen ethyl ester as an ordinate, and obtaining an equation of the ketoprofen ethyl ester, wherein the equation is y =110299x 511.061, and the linear correlation coefficient R =0.9999 of the curve, and the result is shown in Table 1 and FIG. 1.
Table 1: results of the Linear test
2. Specificity test
The detection method of the invention is used for measuring aeration water blank samples, aeration water test sample solutions, algae culture medium blank samples and algae culture medium test sample solutions, and comparing chromatograms of the blank samples and the test samples. The retention time of the ketoprofen ethyl ester is 7.45 minutes, and a blank sample has no interference peak at the time point, so that the result shows that the method has good specificity on the ketoprofen ethyl ester.
3. Repeatability test
The standard working solution with the concentration of 0.200mg/L in example 1 was subjected to continuous sample injection for 6 times to determine the precision, RSD, of the method of the present invention R.T. 0.11% of RSD Area 0.80%, and the results are shown in Table 2.
Table 2: results of precision test
The result shows that the method of the invention meets the requirement on the analysis precision of the same sample.
4. Detection limit and quantification limit
The analysis was performed on the map obtained from the ketoprofen ethyl ester standard working solution of example 1 having a concentration of 0.100mg/L, at which the average signal-to-noise ratio (S/N) =76.39, according to LOD =3 × assay concentration/(S/N), and LOQ =10 × assay concentration/(S/N). The LOD of the present invention was found to be 3.97. Mu.g/L and the LOQ of the present invention was found to be 13.2. Mu.g/L, and the results are shown in Table 3.
Table 3: test results of detection limit and quantification limit
5. Sample application recovery test
Respectively sucking 1.00mL of ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L in example 1 into a 100.0mL volumetric flask, respectively metering the volume to the scale with aerated water and a culture medium, and uniformly mixing to obtain a high-concentration aerated water/culture medium recovery rate test sample with the concentration of 24.0 mg/L. Respectively adding 1.00mL of high-concentration aerated water/culture medium recovery rate test sample into a 100.0mL volumetric flask, fixing the volume to a scale by using the aerated water/culture medium, and uniformly mixing to obtain an aerated water/culture medium low-concentration recovery rate test sample with the concentration of 0.240 mg/L; each concentration sample was made in 5 replicates. Diluting the high concentration recovery rate test sample by 50 times with methanol, directly sampling the low concentration recovery rate sample, measuring with liquid chromatograph, analyzing the concentration of the test substance in the solution, and calculating the recovery rate.
The test was carried out under the operating conditions of the apparatus of example 1, the recovery rate was calculated according to the formula (1), and the Relative Standard Deviation (RSD) of the recovery rate was calculated according to the formulas (2) and (3), and the analysis results are shown in table 4, table 5, fig. 3, fig. 4, fig. 5, and fig. 6,2, in which the average addition recovery rates of ketoprofen ethyl ester at different concentrations in aerated water were 97.1% and 95.4%, respectively, and the relative standard deviation of the recovery rates were 0.45% and 1.03%, respectively; the average addition recovery rate of the 2 ketoprofen ethyl esters with different concentrations in the algae culture medium is 88.6 percent and 94.4 percent respectively, and the relative standard deviation of the recovery rates is 0.31 percent and 1.35 percent respectively.
In the formula:
r-recovery,%;
C d -the measured concentration of the target, mg/L;
in the formula:
s-standard deviation;
xi-recovery,%, from the ith measurement;
n-the number of recovery rates involved in the calculation.
In the formula:
RSD-relative standard deviation,%.
Table 4: test result of recovery rate in aerated water
Table 5: recovery in algal culture medium
6. Stability test
Respectively and accurately weighing 0.1000g of ketoprofen ethyl and 0.1001g of ketoprofen ethyl into a 100.0mL volumetric flask, respectively adding aerated water and an algae culture medium to a constant volume to scale, magnetically stirring for 24 hours in a constant temperature incubator at 25 ℃, and filtering through a 0.45-micrometer filter membrane to obtain a ketoprofen ethyl saturated daphnia acute activity inhibition test stable stock solution and an algae growth inhibition stable stock solution for later use. Weighing 1.08g of ketoprofen ethyl ester in a 1L glass bottle, adding 1L of aerated water, magnetically stirring for 24 hours in a constant temperature incubator at 25 ℃, and filtering to obtain a stable stock solution for the ketoprofen ethyl ester saturated fish acute toxicity test for later use.
The stock solution was diluted 25-fold with methanol, and the concentration of the sample in the solution was analyzed by the detection method of the present invention, and each saturated solution was measured 3 times in parallel.
Taking the average value of the results of the 3 times of measurement as the concentration of the saturated stock solution, and measuring the concentration of the stable stock solution for the ketoprofen ethyl ester saturated daphnia acute activity inhibition test, the concentration of the stable stock solution for the ketoprofen ethyl ester saturated algae growth inhibition test and the concentration of the stable stock solution for the ketoprofen ethyl ester saturated fish acute toxicity test, which are 21.1mg/L, 26.6mg/L and 20.9mg/L.
6.1 stability of daphnia acute activity inhibition test
And adding 30.00mL of saturated daphnia acute activity inhibition test stability stock solution into a 100.0mL volumetric flask, diluting to a constant volume to a scale with aerated water, and uniformly mixing to obtain a daphnia acute activity inhibition test stability sample. Placing the daphnia under the daphnia test condition, sampling at intervals, and diluting by 10 times with methanol; the concentration determination was carried out according to the chromatographic conditions of the present invention. The stability of the target in the sample solution in aerated water was calculated according to the formula (4). The stability results show that the ketoprofen ethyl ester can be maintained at more than 80% of the initial concentration within 48 hours under the daphnia acute activity inhibition test conditions, and the results are shown in table 6, fig. 7 and fig. 8.
The stability of the composite material is improved,% = (Cnh/C0 h) × 100%.. -%
In the formula:
c0h: is the actual concentration of the target at 0 hour;
cn h: is the measured concentration of the target at n hours.
Table 6: stability result of daphnia acute activity inhibition test
6.2 stability of acute toxicity test for Fish
And putting 900mL of the saturated fish acute toxicity test stability stock solution into a 5L beaker, adding 2100mL of aerated water, and uniformly mixing to obtain a fish acute toxicity test stability sample. Sampling at intervals under the condition of fish test, and diluting by 10 times with methanol; the concentration determination was carried out according to the chromatographic conditions of the present invention. The stability of the target in the sample solution in aerated water was calculated according to the formula (4). The stability results show that ketoprofen ethyl ester can be maintained at more than 80% of the initial concentration within 96h under the condition of fish acute toxicity test, and the results are shown in table 7, fig. 9 and fig. 10.
Table 7: stability results of acute toxicity test of fish
6.3 algal growth inhibition test stability
And (3) adding 30.00mL of the stable stock solution for the saturated algae growth inhibition test into a 100.0mL volumetric flask, metering to the scale with a culture medium, and mixing uniformly to obtain a stable sample for the algae growth inhibition test. Placing the sample under the condition of algae test, sampling at intervals, and diluting by 10 times with methanol; the concentration determination was carried out according to the chromatographic conditions of the present invention. The stability of the target in the sample solution in aerated water was calculated according to the formula (4). The stability results show that ketoprofen ethyl ester can be maintained at 80% or more of the initial concentration within 72h under the algae growth inhibition test conditions, and the results are shown in table 8, fig. 11 and fig. 12.
Table 8: stability parallel design of algae production inhibition test
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. An HPLC detection method for ketoprofen ethyl ester in water and algae culture medium is characterized in that a high performance liquid chromatograph and a PDA detector are adopted, and a ZORBAX SB-C18, 250mm x 4.6mm and 5 μm chromatographic column is adopted for detecting the ketoprofen ethyl ester, and the HPLC detection adopts the following chromatographic conditions: the mobile phase A is methanol, and the mobile phase B is water; the flow rate is 0.8-1.2 mL/min; the temperature of the chromatographic column is 30-40 ℃; the detection wavelength is 254nm, and the sample injection amount is 25-35 mu L.
2. The detection method according to claim 1, wherein the volume ratio of the mobile phase is: a: B = 75.
3. The detection method according to claim 1, wherein the flow rate of the mobile phase is 1.0mL/min.
4. The method of claim 1, wherein the column temperature is 32 to 38 ℃.
5. The method of claim 4, wherein the column temperature is 35 ℃.
6. The detection method according to claim 1, wherein the sample volume is 30 μ L.
7. The detection method according to any one of claims 1 to 6, characterized in that it comprises the steps of:
1) Preparing a test solution: weighing 0.242g of ketoprofen ethyl ester sample in a 100.0mL volumetric flask, dissolving the sample with methanol, fixing the volume to the scale, shaking up to obtain a ketoprofen ethyl ester sample stock solution with the concentration of 2.40g/L, taking an appropriate amount of the sample stock solution, diluting the sample stock solution with a diluent to obtain a sample solution, and waiting for HPLC (high performance liquid chromatography) determination;
2) Preparation of standard solution: weighing 0.10081g ketoprofen ethyl ester standard sample in a 100.0mL volumetric flask, dissolving with methanol, fixing the volume to scale, shaking up, and preparing to obtain 1000mg/L ketoprofen ethyl ester standard stock solution; taking and sucking a proper amount of standard stock solution, diluting the stock solution into a series of standard working solutions with the concentrations of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L, 1.00mg/L and 2.00mg/L by using methanol, and waiting for HPLC determination;
3) Measurement and calculation: and setting instrument parameters, performing HPLC (high performance liquid chromatography) determination on the test sample solution and the standard sample solution after the instrument is stabilized, and calculating the content of the ketoprofen ethyl ester according to an external standard method.
8. The detection method according to claim 7, wherein in the step 1), the diluent is aerated water or an algal culture medium.
9. The detection method according to claim 1, wherein the retention time of ketoprofen ethyl ester is 7.45min.
10. The detection method according to claim 1, wherein the LOD of ketoprofen ethyl ester in the method is 3.97 μ g/L, and the LOQ is 13.2 μ g/L.
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