HPLC Detection Method for Ethyl Ketoprofen in a Water and Algae Culture Medium

Abstract

The present invention discloses a high performance liquid chromatography detection method for ethyl ketoprofen in water and algal culture medium. A ZORBAX SB-C18 chromatographic column is used, and water and methanol are used as the mobile phase with a flow rate of 0.9 - 1.2 mL / min. The column temperature is 30 - 40 °C, and the detection wavelength is 254 nm. The average spiked recovery rate of ethyl ketoprofen in aerated water by this method is 95.4% - 97.1%, and the average spiked recovery rate in algal culture medium is 88.6% - 94.4%. The present invention realizes the effective detection of ethyl ketoprofen, and the detection results have good accuracy, high sensitivity, simple operation, low cost, short analysis time and good repeatability, providing a guarantee for the quality control of ethyl ketoprofen.

Description

Technical Field

[0001] The present invention belongs to the field of organic compound analysis, and particularly relates to an HPLC detection method for ethyl ketoprofen in water and algal culture medium. Background Art

[0002] Ethyl ketoprofen, with the chemical name of ethyl 3-benzoyl-α-methylphenylacetate and the molecular formula of C 18 H 18 O3, is often used as an intermediate of ketoprofen to synthesize ketoprofen and its chiral enantiomers. Its structural formula is:

[0003]

[0004] Ketoprofen is a typical non-steroidal anti-inflammatory, antipyretic and analgesic drug, and is widely used in the treatment of diseases such as rheumatoid arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis and gout.

[0005] Chinese Patent ZL201110222136.3, a preparation method of ethyl ketoprofen, discloses a method for preparing ethyl ketoprofen by reacting 2-(3-benzoylphenyl) propionitrile with ethanol in the presence of sulfuric acid, but does not disclose the quality control method of ethyl ketoprofen.

[0006] At present, usually only the conventional physical and chemical indexes of ethyl ketoprofen are detected, and it is difficult to truly reflect its quality status only through the conventional physical and chemical indexes. Therefore, establishing a method that can accurately qualitatively and quantitatively detect ethyl ketoprofen has great guiding significance for its production and application. Summary of the Invention

[0007] The purpose of the present invention is to make up for the deficiencies of the above-mentioned existing technologies, and provide an HPLC detection method for ethyl ketoprofen in water and algal culture medium to more accurately achieve the quality control of ethyl ketoprofen.

[0008] The purpose of the present invention will be achieved through the following technical solutions:

[0009] An HPLC detection method for ethyl ketoprofen in water and algal culture medium, using a high performance liquid chromatograph and a PDA detector, and detecting ethyl ketoprofen with a ZORBAX SB-C18, 250 mm×4.6 mm, 5 μm chromatographic column. The HPLC detection adopts the following chromatographic conditions: mobile phase A is methanol, B is water; the flow rate is 0.8-1.2 mL / min; the column temperature is 30-40 °C; the detection wavelength is 254 nm, and the injection volume is 25-35 μL.

[0010] The volume ratio of the mobile phase methanol to water is 75:25.

[0011] Preferably, the flow rate of the mobile phase is 1.0 mL / min.

[0012] Preferably, the temperature of the chromatographic column is 32 - 38 °C.

[0013] More preferably, the temperature of the chromatographic column is 35 °C.

[0014] Preferably, the sample injection volume is 30 μL.

[0015] The detection method comprises the following steps: (1) Preparation of the test solution: Weigh 0.242 g of ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with the diluent to obtain the test solution for HPLC determination. (2) Preparation of the standard solution: Weigh 0.10081 g of ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a standard stock solution of ethyl ketoprofen with a concentration of 1000 mg / L. Take an appropriate amount of the standard stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination. (3) Determination and calculation: Set the instrument parameters. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0016] Preferably, the diluent is aerated water or algal culture medium.

[0017] Under the chromatographic conditions of this method, the retention time of ethyl ketoprofen is 7.45 min.

[0018] The LOD of this method for ethyl ketoprofen is 3.97 μg / L, and the LOQ is 13.2 μg / L.

[0019] Beneficial effects

[0020] 1. The present invention has established an HPLC detection method for ethyl ketoprofen in water and algal culture medium, overcoming the problem that the quality control of ethyl ketoprofen in the prior art is still blank, and providing a reference for the quality control in the production and application process of ethyl ketoprofen and its related research processes. The method of the present invention has been verified by experimental studies, showing strong specificity, good repeatability, good linear relationship, and strong stability, and can meet the quality control requirements of ethyl ketoprofen.

[0021] 2. The linearity test of the present invention shows that a good linear relationship exists in the range of 0.100 mg / L to 2.00 mg / L. The linear equation is y = 110299x + 511.061, and the linear correlation coefficient R of this curve is 0.9999.

[0022] 3. By comparing the spectra of the aerated water, the blank sample of the algal culture medium, and the sample with added recovery rate, ethyl ketoprofen peaks at 7.45 min, and there is no interfering peak at this time point in the blank sample. The results show that this method has good specificity for ethyl ketoprofen.

[0023] 4. For the target peak of this method, RSDR.T is 0.11% and RSDArea is 0.80%, both of which are less than 5.00%. The results show that this method can be used for the qualitative and quantitative analysis of ethyl ketoprofen; the LOD of this method is 3.97 μg / L; the LOQ is 13.2 μg / L, which can meet the detection requirements.

[0024] 5. The recovery test of the present invention shows that the average added recovery rates of ethyl ketoprofen at different concentrations in aerated water are 95.4% - 97.1%, and the average added recovery rates in the algal culture medium are 88.6% - 94.4%.

[0025] 6. The stability test of the present invention shows that the detection and analysis method of the present invention can respectively meet the concentration determination requirements of the acute activity inhibition test of daphnia, the algal growth inhibition test, and the acute toxicity test of fish. Description of the Drawings

[0026] Figure 1 It is a linear fitting curve graph of ethyl ketoprofen;

[0027] Figure 2 It is a typical spectrum of ethyl ketoprofen;

[0028] Figure 3 It is a typical spectrum of the recovery spiked solution of ethyl ketoprofen at a low concentration in aerated water;

[0029] Figure 4 It is a typical spectrum of the recovery spiked solution of ethyl ketoprofen at a high concentration in aerated water;

[0030] Figure 5 It is a typical spectrum of the recovery spiked solution of ethyl ketoprofen at a low concentration in the algal culture medium;

[0031] Figure 6 It is a typical spectrum of the recovery spiked solution of ethyl ketoprofen at a low concentration in the algal culture medium;

[0032] Figure 7 It is a stability spectrum of ethyl ketoprofen in the fish test environment (0 h);

[0033] Figure 8It is the stability diagram (96h) of ethyl ketoprofen in the fish test environment;

[0034] Figure 9 It is the stability diagram (0h) of ethyl ketoprofen in the daphnia test environment;

[0035] Figure 10 It is the stability diagram (48h) of ethyl ketoprofen in the daphnia test environment;

[0036] Figure 11 It is the stability diagram (0h) of ethyl ketoprofen in the algae test environment;

[0037] Figure 12 It is the stability diagram (72h) of ethyl ketoprofen in the algae test environment. Specific implementation manners

[0038] The present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. The reagents and instruments used in the embodiments that are not indicated by the manufacturer can be obtained as conventional products through commercial purchase. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0039] The instruments and reagents involved in the following embodiments include but are not limited to:

[0040] Instruments and reagents used in the embodiments:

[0041] 1. Reagents and solvents

[0042] (1) Methanol: HPLC, Shanghai Anpu Experimental Technology Co., Ltd.;

[0043] (2) UP water: Resistivity, 18.2 MΩ×cm;

[0044] (3) Aerated water: Self-made in the laboratory;

[0045] (4) Algae medium: Self-made in the laboratory;

[0046] (5) Ethyl ketoprofen, with a purity of 99.2%.

[0047] 2. Main instrument equipment

[0048] (1) High-performance liquid chromatograph: Shimadzu LC2030C 3D Plus;

[0049] (2) SHIMAD LabSolutions data acquisition, qualitative analysis, and quantitative analysis workstation;

[0050] (3) Electronic balance: METTLER TOLEDO (China) Co., Ltd., XSE205D, UEL204;

[0051] (4) Biochemical incubator, ZXSD - B1430.

[0052] Example 1

[0053] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB - C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); the flow rate is 1.0 mL / min; the column temperature is 35 °C; the detection wavelength is 254 nm, and the injection volume is 30 μL.

[0054] (2) Preparation of test solution: Weigh 0.242 g of ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with aerated water to obtain the test solution, and determine it according to the HPLC chromatographic conditions;

[0055] (3) Preparation of standard solution: Weigh 0.10081 g of ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a standard stock solution of ethyl ketoprofen with a concentration of 1000 mg / L; Take an appropriate amount of the standard stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0056] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0057] Example 2

[0058] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB - C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); the flow rate is 0.8 mL / min; the column temperature is 30 °C; the detection wavelength is 254 nm, and the injection volume is 25 μL.

[0059] (2) Preparation of the test solution: Weigh 0.242 g of ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with aerated water to obtain the test solution, and determine it according to the HPLC chromatographic conditions;

[0060] (3) Preparation of the standard solution: Weigh 0.10081 g of ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a standard stock solution of ethyl ketoprofen with a concentration of 1000 mg / L; Take an appropriate amount of the standard stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0061] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0062] Example 3

[0063] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; The mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); The flow rate is 0.9 mL / min; The column temperature is 32 °C; The detection wavelength is 254 nm, and the injection volume is 28 μL.

[0064] (2) Preparation of the test solution: Weigh 0.242 g of ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with aerated water to obtain the test solution, and determine it according to the HPLC chromatographic conditions;

[0065] (3) Preparation of the standard solution: Weigh 0.10081 g of ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a standard stock solution of ethyl ketoprofen with a concentration of 1000 mg / L; Take an appropriate amount of the standard stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0066] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0067] Example 4

[0068] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); the flow rate is 1.1 mL / min; the column temperature is 38 °C; the detection wavelength is 254 nm, and the injection volume is 32 μL.

[0069] (2) Preparation of the test solution: Weigh 0.242 g of the ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with aerated water to obtain the test solution, and determine it according to the HPLC chromatographic conditions;

[0070] (3) Preparation of the standard solution: Weigh 0.10081 g of the ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a standard stock solution of ethyl ketoprofen with a concentration of 1000 mg / L; Take an appropriate amount of the standard stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0071] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0072] Example 5

[0073] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); the flow rate is 1.2 mL / min; the column temperature is 40 °C; the detection wavelength is 254 nm, and the injection volume is 35 μL.

[0074] (2) Preparation of the test solution: Weigh 0.242 g of ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with aerated water to obtain the test solution, and determine it according to the HPLC chromatographic conditions;

[0075] (3) Preparation of the standard solution: Weigh 0.10081 g of ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, shake well to prepare a stock solution of ethyl ketoprofen standard with a concentration of 1000 mg / L; Take an appropriate amount of the stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0076] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0077] Example 6

[0078] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; The mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); The flow rate is 1.0 mL / min; The column temperature is 35 °C; The detection wavelength is 254 nm, and the injection volume is 30 μL.

[0079] (2) Preparation of the test solution: Weigh 0.242 g of ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with algal culture medium to obtain the test solution, and determine it according to the HPLC chromatographic conditions;

[0080] (3) Preparation of the standard solution: Weigh 0.10081 g of ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, shake well to prepare a stock solution of ethyl ketoprofen standard with a concentration of 1000 mg / L; Take an appropriate amount of the stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0081] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0082] Example 7

[0083] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); the flow rate is 0.8 mL / min; the column temperature is 30 °C; the detection wavelength is 254 nm, and the injection volume is 25 μL.

[0084] (2) Preparation of the test solution: Weigh 0.242 g of the ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with algal culture medium to obtain the test solution, and perform the determination according to the HPLC chromatographic conditions;

[0085] (3) Preparation of the standard solution: Weigh 0.10081 g of the ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a stock solution of ethyl ketoprofen standard with a concentration of 1000 mg / L; Take an appropriate amount of the stock solution and dilute it with methanol to prepare a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0086] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0087] Example 8

[0088] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); the flow rate is 0.9 mL / min; the column temperature is 32 °C; the detection wavelength is 254 nm, and the injection volume is 28 μL.

[0089] (2) Preparation of the test solution: Weigh 0.242 g of ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with algal culture medium to obtain the test solution, and determine it according to the HPLC chromatographic conditions;

[0090] (3) Preparation of the standard solution: Weigh 0.10081 g of ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a stock solution of ethyl ketoprofen standard with a concentration of 1000 mg / L; Take an appropriate amount of the stock solution and dilute it with methanol to prepare a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0091] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0092] Example 9

[0093] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; The mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); The flow rate is 1.1 mL / min; The column temperature is 38 °C; The detection wavelength is 254 nm, and the injection volume is 32 μL.

[0094] (2) Preparation of the test solution: Weigh 0.242 g of ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with algal culture medium to obtain the test solution, and determine it according to the HPLC chromatographic conditions;

[0095] (3) Preparation of the standard solution: Weigh 0.10081 g of ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a stock solution of ethyl ketoprofen standard with a concentration of 1000 mg / L; Take an appropriate amount of the stock solution and dilute it with methanol to prepare a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0096] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0097] Example 10

[0098] (1) HPLC chromatographic conditions: The chromatographic column is ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is A: methanol, B: water; A:B = 75:25 (v / v); the flow rate is 1.2 mL / min; the column temperature is 40 °C; the detection wavelength is 254 nm, and the injection volume is 35 μL.

[0099] (2) Preparation of the test solution: Weigh 0.242 g of the ethyl ketoprofen sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a stock solution of ethyl ketoprofen sample with a concentration of 2.40 g / L. Take an appropriate amount of the stock solution and dilute it with algal culture medium to obtain the test solution, and perform the determination according to the HPLC chromatographic conditions;

[0100] (3) Preparation of the standard solution: Weigh 0.10081 g of the ethyl ketoprofen standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a standard stock solution of ethyl ketoprofen with a concentration of 1000 mg / L; Take an appropriate amount of the standard stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination;

[0101] (4) Determination and calculation: Set the instrument parameters according to the HPLC chromatographic conditions. After the instrument is stable, perform HPLC determination on the test solution and the standard solution, and calculate the content of ethyl ketoprofen by the external standard method.

[0102] To further confirm the feasibility of the present invention, the inventor verified the detection method of the present invention through a large number of analytical method tests, and some test contents are excerpted as follows:

[0103] 1. Linear test

[0104] Precisely pipette 1.00 mL of the standard working solutions with a series of concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L in Example 1, and perform high-performance liquid chromatography determination according to the detection method of the present invention. Using the concentration of ethyl ketoprofen as the abscissa and the peak area of ethyl ketoprofen as the ordinate, plot a calibration curve. The equation of ethyl ketoprofen is y = 110299x + 511.061, and the linear correlation coefficient R of this curve is 0.9999. The results are shown in Table 1 and Figure 1 。

[0105] Table 1: Results of the linearity test

[0106]

[0107]

[0108] 2. Specificity test

[0109] Use the detection method of the present invention to measure the blank sample of aerated water, the test solution of aerated water, the blank sample of algal culture medium, and the test solution of algal culture medium, and compare the chromatograms of the blank sample and the test solution. The retention time of ethyl ketoprofen is 7.45 minutes, and there is no interference peak in the blank sample at this time point. The results show that the specificity of this method for ethyl ketoprofen is good.

[0110] 3. Repeatability test

[0111] Inject the standard working solution with a concentration of 0.200 mg / L in Example 1 continuously for 6 times to measure the precision of the method of the present invention. The RSD R.T. is 0.11%, and the RSD Area is 0.80%. The results are shown in Table 2.

[0112] Table 2: Results of the precision test

[0113]

[0114]

[0115] The results show that the precision of the method of the present invention for the analysis of the same sample meets the requirements.

[0116] 4. Detection limit and quantification limit

[0117] The spectrum obtained from the ketoprofen ethyl ester standard working solution with a concentration of 0.100 mg / L in Example 1 was analyzed. At this concentration, the average signal-to-noise ratio (S / N) = 76.39. According to LOD = 3 × analytical concentration / (S / N) and LOQ = 10 × analytical concentration / (S / N), the LOD of the present invention was 3.97 μg / L and the LOQ was 13.2 μg / L. The results are shown in Table 3.

[0118] Table 3: Test results of detection limit and quantification limit

[0119]

[0120] 5. Spike recovery test

[0121] 1.00 mL of the ketoprofen ethyl ester sample stock solution with a concentration of 2.40 g / L in Example 1 was respectively pipetted into 100.0 mL volumetric flasks, and diluted to the mark with aerated water and culture medium respectively, and mixed well to obtain high-concentration aerated water / culture medium recovery test samples with a concentration of 24.0 mg / L. 1.00 mL of the high-concentration aerated water / culture medium recovery test samples was respectively added to 100.0 mL volumetric flasks, and diluted to the mark with aerated water / culture medium and mixed well to obtain low-concentration aerated water / culture medium recovery test samples with a concentration of 0.240 mg / L; 5 replicates were prepared for each concentration sample. The high-concentration recovery test samples were diluted 50 times with methanol, and the low-concentration recovery samples were directly sampled. The concentration of the test substance in the solution was determined by liquid chromatograph and the recovery rate was calculated.

[0122] The tests were carried out according to the instrument operating conditions in Example 1. The recovery rate was calculated according to formula (1), and the relative standard deviation (RSD) of the recovery rate was calculated according to formula (2) and formula (3). The analysis results are shown in Table 4, Table 5, Figure 3 、 Figure 4 、 Figure 5 and Figure 6 , the average spike recoveries of two different concentrations of ketoprofen ethyl ester in aerated water were 97.1% and 95.4% respectively, and the relative standard deviations of the recovery rates were 0.45% and 1.03% respectively; the average spike recoveries of two different concentrations of ketoprofen ethyl ester in algal culture medium were 88.6% and 94.4% respectively, and the relative standard deviations of the recovery rates were 0.31% and 1.35% respectively.

[0123]

[0124] Where:

[0125] R - recovery rate, %;

[0126] C d —— measured concentration of the target substance, mg / L;

[0127]

[0128] In the formula:

[0129] S——Standard deviation;

[0130] Xi——Recovery rate obtained from the i-th measurement, %;

[0131] ——Average value of the recovery rate, %;

[0132] N——Number of recovery rates involved in the calculation.

[0133]

[0134] In the formula:

[0135] RSD——Relative standard deviation, %.

[0136] Table 4: Results of the recovery rate test in aerated water

[0137]

[0138] Table 5: Recovery rate in algal culture medium

[0139]

[0140] 6. Stability test

[0141] Accurately weigh 0.1000 g and 0.1001 g of ethyl ketoprofen into 100.0 mL volumetric flasks respectively, add aerated water and algal culture medium respectively to make up to the mark, stir magnetically in a constant temperature incubator at 25 °C for 24 h, filter through a 0.45 μm filter membrane to obtain the stability stock solutions of ethyl ketoprofen for the acute activity inhibition test of daphnia and the stability stock solution of algal growth inhibition for standby. Weigh 1.08 g of ethyl ketoprofen into a 1 L glass bottle, add 1 L of aerated water, stir magnetically in a constant temperature incubator at 25 °C for 24 h, filter to obtain the stability stock solution of ethyl ketoprofen for the acute toxicity test of fish for standby.

[0142] Take the stock solution and dilute it 25 times with methanol, measure it by the detection method of the present invention, analyze the concentration of the test substance in the solution, and measure each saturated solution in parallel 3 times.

[0143] Take the average value of the 3 measurement results as the concentration of the saturated stock solution. After measurement, the concentration of the stability stock solution of ethyl ketoprofen for the acute activity inhibition test of daphnia is 21.1 mg / L, the concentration of the stability stock solution of ethyl ketoprofen for the algal growth inhibition stability is 26.6 mg / L, and the concentration of the stability stock solution of ethyl ketoprofen for the acute toxicity test of fish is 20.9 mg / L.

[0144] 6.1 Stability of the acute immobilization test of Daphnia

[0145] Take 30.00 mL of the saturated stock solution for the stability of the acute immobilization test of Daphnia and add it to a 100.0 mL volumetric flask. Dilute to the mark with aerated water and mix evenly to obtain a sample for the stability of the acute immobilization test of Daphnia. Place it under the Daphnia test conditions, take samples at intervals, and dilute 10 times with methanol; determine the concentration according to the chromatographic conditions of the present invention. Calculate the stability of the target substance in aerated water in this sample solution according to formula (4). The stability results show that ethyl ketoprofen can maintain above 80% of the initial concentration within 48 h under the conditions of the acute immobilization test of Daphnia. The results are shown in Table 6, Figure 7 , Figure 8 .

[0146] Stability, % = (Cnh / C0h) × 100%..............(4)

[0147] In the formula:

[0148] C0h: is the measured concentration of the target substance at 0 h;

[0149] Cnh: is the measured concentration of the target substance at n h.

[0150] Table 6: Results of the stability of the acute immobilization test of Daphnia

[0151]

[0152] 6.2 Stability of the acute toxicity test of fish

[0153] Take 900 mL of the saturated stock solution for the stability of the acute toxicity test of fish and place it in a 5 L beaker. Add 2100 mL of aerated water and mix evenly to obtain a sample for the stability of the acute toxicity test of fish. Place it under the fish test conditions, take samples at intervals, and dilute 10 times with methanol; determine the concentration according to the chromatographic conditions of the present invention. Calculate the stability of the target substance in aerated water in this sample solution according to formula (4). The stability results show that ethyl ketoprofen can maintain above 80% of the initial concentration within 96 h under the conditions of the acute toxicity test of fish. The results are shown in Table 7, Figure 9 , Figure 10 .

[0154] Table 7: Results of the stability of the acute toxicity test of fish

[0155]

[0156]

[0157] 6.3 Stability of the algal growth inhibition test

[0158] Take 30.00 mL of the stability stock solution for the algal growth inhibition test and add it to a 100.0 mL volumetric flask. Dilute it to the mark with the culture medium and mix well to obtain the stability sample for the algal growth inhibition test. Place it under the algal test conditions, sample at intervals, and dilute it 10 times with methanol; determine the concentration according to the chromatographic conditions of the present invention. Calculate the stability of the target substance in the aerated water in the sample solution according to formula (4). The stability results show that ethyl ketoprofen can maintain more than 80% of the initial concentration within 72 h under the conditions of the algal growth inhibition test. The results are shown in Table 8, Figure 11 , Figure 12 .

[0159] Table 8: Design of the stability parallel for the algal production inhibition test

[0160]

[0161] Although the present invention has been described in detail above with general descriptions, specific embodiments and tests, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. An HPLC detection method for ethyl ketoprofen in a water and algae culture medium, characterized in that, Using a high performance liquid chromatograph and a PDA detector, ketoprofen ethyl ester was detected with a ZORBAX SB-C18, 250 mm×4.6 mm, 5 μm chromatographic column. The HPLC detection was carried out under the following chromatographic conditions: mobile phase A was methanol, and B was water; the flow rate was 0.8 - 1.2 mL / min; the column temperature was 30 - 40 °C; the detection wavelength was 254 nm, and the injection volume was 25 - 35 μL; The detection method includes the following steps: 1) Preparation of the test solution: Weigh 0.242 g of ketoprofen ethyl ester sample into a 100.0 mL volumetric flask, dissolve it with methanol and make up to the mark, shake well to obtain a ketoprofen ethyl ester sample stock solution with a concentration of 2.40 g / L. Take an appropriate amount of the sample stock solution and dilute it with a diluent to obtain the test solution for HPLC determination; 2) Preparation of the standard solution: Weigh 0.10081 g of ketoprofen ethyl ester standard into a 100.0 mL volumetric flask, dissolve it with methanol, make up to the mark, and shake well to prepare a ketoprofen ethyl ester standard stock solution with a concentration of 1000 mg / L. Take an appropriate amount of the standard stock solution and dilute it with methanol to a series of standard working solutions with concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, 1.00 mg / L, and 2.00 mg / L for HPLC determination; 3) Determination and calculation: Set the instrument parameters. After the instrument is stable, carry out HPLC determination on the test solution and the standard solution, and calculate the content of ketoprofen ethyl ester by the external standard method; In step 1), the diluent is aerated water or algal culture medium; The volume ratio of the mobile phase is: A:B = 75:

25.

2. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase is 1.0 mL / min.

3. The detection method according to claim 1, characterized in that The column temperature is 32 - 38 °C.

4. The detection method according to claim 3, wherein, The column temperature is 35 °C.

5. The detection method according to claim 1, wherein The injection volume is 30 μL.

6. The detection method according to claim 1, wherein The retention time of ketoprofen ethyl ester is 7.45 min.

7. The detection method according to claim 1, wherein The LOD of this method for ketoprofen ethyl ester is 3.97 μg / L, and the LOQ is 13.2 μg / L.

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