CN110954392A - Method for detecting enzyme protein residue in cefprozil prepared by enzyme method - Google Patents
Method for detecting enzyme protein residue in cefprozil prepared by enzyme method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 67
- WDLWHQDACQUCJR-ZAMMOSSLSA-N (6r,7r)-7-[[(2r)-2-azaniumyl-2-(4-hydroxyphenyl)acetyl]amino]-8-oxo-3-[(e)-prop-1-enyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)/C=C/C)C(O)=O)=CC=C(O)C=C1 WDLWHQDACQUCJR-ZAMMOSSLSA-N 0.000 title claims abstract description 56
- 229960002580 cefprozil Drugs 0.000 title claims abstract description 56
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 72
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 claims abstract description 67
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 36
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 36
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 22
- 239000012488 sample solution Substances 0.000 claims description 20
- 238000002156 mixing Methods 0.000 claims description 19
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
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- 229940079593 drug Drugs 0.000 abstract description 7
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- 229930182555 Penicillin Natural products 0.000 description 10
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 229940049954 penicillin Drugs 0.000 description 10
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- 206010067484 Adverse reaction Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010033078 Otitis media Diseases 0.000 description 1
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 1
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- 239000003960 organic solvent Substances 0.000 description 1
- -1 p-hydroxy-phenyl Chemical group 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
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- 229910021642 ultra pure water Inorganic materials 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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Abstract
The invention provides a method for detecting enzyme protein residue in cefprozil prepared by an enzyme method, which is characterized by comprising the following steps: comprises preparing Coomassie brilliant blue G-250 dye solution and standard protein stock solution; preparing a standard curve solution by using a sodium bicarbonate solution as a solvent; preparing a blank solution by taking a sodium bicarbonate solution as a solvent; preparing a test sample determination solution by using a sodium bicarbonate solution as a solvent; using an ultraviolet spectrophotometer, and drawing a standard curve of absorbance-protein concentration through blank solution correction; and calculating the residual content of the enzyme protein contained in the test sample according to the absorbance value read by the test sample determination solution. According to the invention, sodium bicarbonate is skillfully selected as a solvent, cefprozil can be dissolved, meanwhile, the zymoprotein is fully dissolved, the residual quantity of the zymoprotein in the cefprozil product prepared by the enzyme method can be effectively measured, the drug use safety of the drug is improved, and the detection method is simple to operate, high in accuracy, and good in reproducibility and reliability.
Description
Technical Field
The invention relates to the field of pharmacy, and in particular relates to a method for detecting enzyme protein residues in cefprozil prepared by an enzyme method.
Background
Cefprozil (Cefprozil) has the chemical name of (6R,7R) -7- [ (R) -2-amino-2- (p-hydroxy-phenyl) acetamido ] -8-oxo-3-propene-5-thia-1-azabicyclo [4,2,0] oct-2-ene-2-carboxylic acid-hydrate, is the second generation of non-ester oral cephalosporin broad-spectrum antibacterial drug developed by Betamet-Spanish GmbH, USA, and is the first oral cephalosporin antibiotic approved by FDA for treating otitis media and sinusitis in children.
At present, the reported method for synthesizing cefprozil mainly adopts a chemical synthesis method, and in recent years, a new green and environment-friendly preparation process for synthesizing cefprozil by an enzyme method is also reported. Compared with chemical synthesis, the enzymatic synthesis has the advantages of less used organic solvent and simple route, can overcome the defects of high cost, large use of toxic reagents and long production period of the existing chemical synthesis method, and meets the requirements of economic, environmental and social progress.
However, in the process for enzymatically synthesizing cefprozil, a synthetase is used, and the structure of the synthetase is mainly protein. If a large amount of the protein remains in the finished product, it is an exogenous protein for human body after administration, and may cause adverse reactions such as allergy to the patient. Therefore, in order to better detect the quality of cefprozil drugs prepared by the enzymatic method, it is necessary to provide a detection method for detecting the residual amount of enzyme protein in cefprozil final products prepared by the enzymatic method, thereby ensuring the safety of medication.
Disclosure of Invention
The invention aims to provide a method for detecting the enzyme protein in cefprozil prepared by an enzyme method, which has the advantages of simple operation, high accuracy, good reproducibility and reliability, can effectively measure the residual quantity of the enzyme protein in cefprozil products prepared by the enzyme method, and improves the medication safety of medicines.
The invention is realized by the following technical scheme:
a detection method for enzyme protein residue in cefprozil prepared by an enzyme method is characterized by comprising the following steps:
preparing Coomassie brilliant blue G-250 dye solution and standard protein stock solution;
preparation of standard curve solution: taking standard protein stock solutions, and diluting with sodium bicarbonate solutions with the same concentration and different volumes respectively to obtain respective standard protein linear solutions; respectively adding the Coomassie brilliant blue G-250 dye solution with the same volume into the standard protein linear solutions, and uniformly mixing; standing for reaction to obtain a standard curve solution;
preparation of a blank solution: taking Coomassie brilliant blue G-250 dye solution with the same volume as that of the standard curve solution during preparation, adding sodium bicarbonate solution with the same concentration, mixing uniformly, standing for reaction, and taking the mixture as blank solution;
preparation of test article assay solution: weighing a proper amount of cefprozil test sample, adding sodium bicarbonate solution with the same concentration for dissolving and diluting to obtain test sample solution; adding the Coomassie brilliant blue G-250 dye solution with the same volume as that of the standard curve solution during preparation into the test solution, mixing uniformly, and standing for reaction to obtain a test solution;
using an ultraviolet spectrophotometer, correcting through a blank solution, determining absorbance by adopting a standard curve solution, and drawing a standard curve according to the concentration and absorbance value of each protein linear solution in the standard curve solution; and calculating the protein concentration in the test solution of the test sample by adopting a standard curve method according to the absorbance value read by the test solution of the test sample, thereby calculating the residual content of the enzyme protein contained in the test sample.
The existing detection method for protein content mostly selects water as a diluent, and cefprozil is slightly soluble in water due to the dissolution characteristic, so that the enzyme protein residue in cefprozil cannot be quantitatively determined by adopting the conventional detection method for protein content; the invention skillfully selects the sodium bicarbonate as the solvent, can dissolve the cefprozil, simultaneously fully dissolves the zymoprotein, does not cause the damage of the zymoprotein due to the denaturation of the zymoprotein, and enables the detection of the residual zymoprotein in the cefprozil to be possible, and the detection method is reasonable, simple to operate, high in accuracy, good in reproducibility and reliability.
Further, the method for detecting the enzyme protein residue in the cefprozil prepared by the enzyme method is characterized by comprising the following steps: further comprising the steps of:
preparation of Coomassie brilliant blue G-250 dye solution: weighing Coomassie brilliant blue G-250, dissolving in ethanol, adding appropriate amount of phosphoric acid, and diluting to constant volume with purified water;
preparation of standard protein stock solution: weighing a proper amount of bovine serum albumin, and dissolving and diluting the bovine serum albumin with purified water to obtain a standard protein stock solution.
As an embodiment, in the preparation process of the coomassie brilliant blue G-250 dye solution, the ratio of the weight of the coomassie brilliant blue G-250 to the volume of ethanol to the volume of phosphoric acid to the volume of constant volume is 5G: (2-3) L: (5-6) L: 50L.
In one embodiment, the standard protein stock solution has a concentration of 0.1-5 mg/ml, and the sample solution has a concentration of 5-100 mg/ml. The preparation of the concentration of the standard protein stock solution can correspond to the concentration of the test solution, so that the accuracy of the detection method is further improved.
Preferably, the concentration of the sodium bicarbonate solution is 1% to 15% (g/ml). If the concentration of the sodium bicarbonate solution is too low (less than 1% g/ml), cefprozil cannot be dissolved; if the concentration of the sodium bicarbonate solution is too high (above 15% g/ml), damage to the protein may result.
Preferably, the standing reaction time in the preparation of the blank solution and the standing reaction time in the preparation of the standard curve solution are the same as the standing reaction time in the preparation of the test sample measurement solution. The reaction degree of the Coomassie brilliant blue G-250 and the protein is equivalent, and the accuracy of the detection method is improved; the volume of the standard protein linear solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the standard curve solution, the volume of the test sample solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the test sample determination solution and the volume of the sodium bicarbonate solution added during the preparation of the blank solution are equal. The above conditions further improve the accuracy of the detection method.
Further, the standing reaction time during the preparation of the blank solution, the standing reaction time during the preparation of the standard curve solution and the standing reaction time during the preparation of the test sample determination solution are all 2-20 minutes. If the reaction time is less than 2 minutes, the reaction may not occur, and if it is more than 20 minutes, precipitation may occur to affect the detection result. Preferably, the selected wavelength range is 590-600 nm by using the ultraviolet spectrophotometer.
Preferably, at least five standard protein linear solutions are prepared, which can improve the accuracy of the standard curve and the accuracy of the final detection result.
As an embodiment, the method for detecting enzyme protein residue in cefprozil prepared by the enzymatic method is characterized in that:
the preparation method of the Coomassie brilliant blue G-250 dye solution comprises the following steps: weighing 100mg of Coomassie brilliant blue G-250, dissolving in 40-60 ml of ethanol, adding 120ml of phosphoric acid 100 and adding purified water to a constant volume of 1000 ml;
the standard protein stock solution has the concentration of 0.1-5 mg/ml, and the standard protein linear solution has the following concentrations: 25 mu g/ml, 100 mu g/ml, 200 mu g/ml, 400 mu g/ml, 600 mu g/ml, 800 mu g/ml and 1000 mu g/ml, wherein the concentration c of the sodium bicarbonate solution is 1-15% (g/ml);
the concentration of the test solution is 5-100 mg/ml;
the standing reaction time during the preparation of the blank solution, the standing reaction time during the preparation of the standard curve solution and the standing reaction time during the preparation of the test sample determination solution are all 2-20 minutes, the volumes of the Coomassie brilliant blue G-250 dye solutions adopted by the standard curve solution, the blank solution and the test sample determination solution are all 5-25 ml, and the volumes of the standard protein linear solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the standard curve solution, the test sample solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the test sample determination solution and the sodium bicarbonate solution added during the preparation of the blank solution are all 10-250 muL.
According to the detection method for preparing the enzyme protein in the cefprozil by the enzyme method, sodium bicarbonate is skillfully selected as a solvent, so that the cefprozil can be dissolved, the enzyme protein can be fully dissolved, the enzyme protein is not damaged due to the denaturation of the protein of the enzyme protein, the detection of the residual enzyme protein in the cefprozil which is slightly dissolved in water becomes possible, and the detection method is reasonable, simple to operate, high in accuracy, good in reproducibility and reliability; the residual quantity of the zymoprotein in the cefprozil product prepared by the enzyme method can be effectively measured, and the medication safety of the medicine is improved.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a graph of a practical standard curve of example 1 of the present invention;
FIG. 2 is a graph of a standard curve for analysis reproducibility in example 1 of the present invention;
FIG. 3 is a graph showing the results of the calibration of the intermediate precision in example 1 of the present invention.
Detailed Description
The method for detecting the enzyme protein residue in the cefprozil prepared by the enzyme method comprises the following steps:
preparing Coomassie brilliant blue G-250 dye solution and standard protein stock solution;
preparation of standard curve solution: taking standard protein stock solutions, and diluting with sodium bicarbonate solutions with the same concentration and different volumes respectively to obtain respective standard protein linear solutions; respectively adding the Coomassie brilliant blue G-250 dye solution with the same volume into the standard protein linear solutions, and uniformly mixing; standing for reaction to obtain a standard curve solution;
preparation of a blank solution: taking Coomassie brilliant blue G-250 dye solution with the same volume as that of the standard curve solution during preparation, adding sodium bicarbonate solution with the same concentration, mixing uniformly, standing for reaction, and taking the mixture as blank solution;
preparation of test article assay solution: weighing a proper amount of cefprozil test sample, adding sodium bicarbonate solution with the same concentration for dissolving and diluting to obtain test sample solution; adding the Coomassie brilliant blue G-250 dye solution with the same volume as that of the standard curve solution during preparation into the test solution, mixing uniformly, and standing for reaction to obtain a test solution;
using an ultraviolet spectrophotometer, correcting through a blank solution, determining absorbance by adopting a standard curve solution, and drawing a standard curve according to the concentration and absorbance value of each protein linear solution in the standard curve solution; and calculating the protein concentration in the test solution of the test sample by adopting a standard curve method according to the absorbance value read by the test solution of the test sample, thereby calculating the residual content of the enzyme protein contained in the test sample.
The linear concentrations of the Coomassie brilliant blue G-250 staining solution, the standard protein stock solution, the standard protein linear solution and the test solution are not particularly limited, and can be the conventional concentration in the field so as to be convenient for detection; the volume of the standard protein linear solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the standard curve solution, the volume of the test sample solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the test sample determination solution and the volume of the sodium bicarbonate solution added during the preparation of the blank solution are not particularly limited, and only the three solutions are required to be equal, so that the detection is convenient. In the following examples, bovine serum albumin used to prepare the standard protein stock solution is a commercial product, and coomassie brilliant blue G-250 used in the coomassie brilliant blue G-250 dye solution is a commercial product. Preferably, the standard protein stock solution and the diluent for the test sample are 1% -15% sodium bicarbonate solution to avoid the concentration of the sodium bicarbonate solution being too low to dissolve the protein or too high to cause damage to the protein. The adopted detection equipment is an ultraviolet spectrophotometer, and the detection wavelength is 590-600 nm; the standard curve is established by taking the actual concentration of the protein contained in each linear solution as an abscissa and taking the absorbance obtained by detecting the actual concentration of the protein in the linear solution in an ultraviolet spectrophotometer at a wavelength of 590-600 nm as an ordinate; established standard curve R2Should be usable above 0.9900 otherwise the standard curve should be reproduced.
As an embodiment, preparation of coomassie brilliant blue G-250 dye liquor: weighing Coomassie brilliant blue G-250, dissolving in ethanol, adding appropriate amount of phosphoric acid, and diluting to constant volume with purified water; preparation of standard protein stock solution: weighing a proper amount of bovine serum albumin, and dissolving and diluting the bovine serum albumin with purified water to obtain a standard protein stock solution.
As an embodiment, in the preparation process of the coomassie brilliant blue G-250 dye solution, the ratio of the weight of the coomassie brilliant blue G-250 to the volume of ethanol to the volume of phosphoric acid to the volume of constant volume is 5G: (2-3) L: (5-6) L: 50L.
In one embodiment, the standard protein stock solution has a concentration of 0.1-5 mg/ml, and the sample solution has a concentration of 5-100 mg/ml.
Further, the standing reaction time during the preparation of the blank solution and the standing reaction time during the preparation of the standard curve solution are the same as the standing reaction time during the preparation of the test sample determination solution, and are both 2-20 minutes. If the reaction time is less than 2 minutes, the reaction may not occur, and if it is more than 20 minutes, precipitation may occur to affect the detection result. As a specific embodiment, the method for detecting enzyme protein residues in cefprozil prepared by the enzyme method comprises the following steps: weighing 100mg of Coomassie brilliant blue G-250, dissolving in 40-60 ml of ethanol, adding 120ml of phosphoric acid 100 and adding purified water to a constant volume of 1000 ml; the standard protein stock solution has the concentration of 0.1-5 mg/ml, and the standard protein linear solution has the following concentrations: 25 mu g/ml, 100 mu g/ml, 200 mu g/ml, 400 mu g/ml, 600 mu g/ml, 800 mu g/ml and 1000 mu g/ml, wherein the concentration c of the sodium bicarbonate solution is 1-15% (g/ml); the concentration of the test solution is 5-100 mg/ml; the standing reaction time during the preparation of the blank solution and the standing reaction time during the preparation of the standard curve solution are both 2-20 minutes, the volumes of Coomassie brilliant blue G-250 dye solutions adopted by the standard curve solution, the blank solution and the test article determination solution are both 5-25 ml, and the volumes of standard protein linear solution added into the Coomassie brilliant blue G-250 dye solution, the test article solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the standard curve solution, and the sodium bicarbonate solution added during the preparation of the blank solution are all 10-250 muL.
The following are materials, reagents and instruments used in the following examples.
1. Instrument for measuring the position of a moving object
Ultraviolet spectrophotometer: shimadzu UV-1800
2. Materials, reagents
Bovine serum albumin: biotechnological grade, alatin;
coomassie Brilliant blue G-250: shanghai green bird technical development Co., Ltd;
phosphoric acid: aladdin reagent (Shanghai) Inc.;
ethanol: supplied by Kyoto Biotechnology, Inc., Shanghai;
sodium bicarbonate: guangzhou chemical reagent works;
purifying water: producing Rehille Direct-Pure UP ultrapure water by an integrated machine;
the 5-batch enzymatic synthesis of cefprozil raw material is produced by Baiyunshan chemical pharmaceutical factory, Guangzhou Baiyunshan pharmaceutical group GmbH.
Example 1
The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method comprises the following steps:
1. preparation of test solutions
(1) Preparation of Coomassie brilliant blue G-250 dye solution: weighing 100mg of Coomassie brilliant blue G-250, dissolving in 40-60 ml of ethanol, adding 120ml of phosphoric acid 100 and adding purified water to a constant volume of 1000 ml;
(2) preparation of standard protein stock solution: 125mg of bovine serum albumin was precisely weighed, dissolved in purified water to a volume of 50ml in a volumetric flask, and used as a standard protein stock solution.
(3) Preparation of standard curve solution: taking a standard protein stock solution, diluting with a sodium bicarbonate solution with the concentration of 10% g/ml according to the following table 1, preparing 1 part for each concentration, and obtaining 7 parts of standard protein linear solution in total; respectively taking 10ml of Coomassie brilliant blue G-250 dye solution, respectively placing the dye solution into 7 penicillin bottles, respectively adding 200 mu L of each standard protein linear solution shown in the following table, sealing and uniformly mixing; after standing for 10min, 7 parts of standard curve solution is obtained.
Table 1: theoretical preparation of standard protein Linear solution
In practice, 129.36mg of bovine serum albumin was weighed to prepare the above test solution, and dissolved in purified water to a volume of 50ml in a volumetric flask to obtain a standard protein stock solution with a concentration of 2483.712. mu.g/ml. Taking a proper amount of sodium bicarbonate solution with the concentration of 10% g/ml according to the following table 2, respectively, and diluting to obtain 7 parts of standard protein linear solution; respectively taking 10ml of Coomassie brilliant blue G-250 dye solution, respectively placing the dye solution into 7 penicillin bottles, respectively adding 200 mu L of each standard protein linear solution shown in the following table, sealing and uniformly mixing; after standing for 10min, 7 parts of standard curve solution is obtained.
Table 2: practical preparation of Standard protein Linear solution
| Volume of Standard protein stock solution (mL) | Dilution volume (mL) | Actual concentration (μ g/mL) |
| 1 | 100 | 24.838 |
| 1 | 25 | 99.352 |
| 2 | 25 | 198.705 |
| 4 | 25 | 397.409 |
| 6 | 25 | 596.114 |
| 8 | 25 | 794.819 |
| 10 | 25 | 993.523 |
After the ultraviolet spectrophotometer is used for correcting the blank solution at the wavelength of 595nm, the 7 parts of standard curve solution are respectively put into the ultraviolet spectrophotometer to read the absorbance value, and the detection results are as follows in the following table 3:
table 3: table of absorbance values for each standard curve solution
(4) And preparing a blank solution, namely putting 10ml of Coomassie brilliant blue G-250 dye solution into a penicillin bottle, adding 200 mu L of 10% sodium bicarbonate solution, sealing, uniformly mixing, and standing for 10min to obtain the blank solution.
(5) Preparation of test article assay solution: precisely weighing 500mg of cefprozil test sample, placing the cefprozil test sample in a 10ml volumetric flask, adding 10% sodium bicarbonate solution, ultrasonically dissolving, diluting to a scale, and shaking uniformly to obtain test sample solution; taking 10ml Coomassie brilliant blue G-250 dye solution, placing in a penicillin bottle, adding 200 μ L of test solution, sealing, mixing, standing for 10min, and using as test solution.
2. Establishment of a Standard Curve
Taking a blank solution, and adjusting zero at 595nm by using an ultraviolet spectrophotometer; taking each concentration standard curve solution to respectively measure the absorbance value of each concentration standard curve solution, and taking each linear solutionThe actual concentration of the contained protein is an abscissa (x), the corresponding absorbance value is an ordinate (y), and a standard curve linear regression equation is established: y is kx ± b; the linear regression coefficient R of the established standard curve2Should be above 0.9900.
In actual operation, the obtained actual standard curve is shown in fig. 1, and the linear regression equation is: 0.000538852x +0.0244872, r2The absorbance value LOQ is 0.0244872 at 0.99516.
3. Measurement of test article
Taking a test sample determination solution, and determining the absorbance value of the test sample determination solution by using an ultraviolet spectrophotometer at 595 nm; and (4) obtaining the protein concentration in the test solution of the test sample by applying a standard curve, thereby calculating the residual content of the enzyme protein in the test sample.
Wherein y is the absorbance of the solution measured by the test sample;
b is the intercept of a standard curve linear regression equation; if the absorbance value measured by the test article assay solution is less than b, it is considered to be less than the limit of quantitation (LOQ).
k is the slope of the standard curve linear regression equation;
m is the sample weight of the sample, mu g
v is the dilution volume of the prepared test solution, ml
In actual practice, preparation of a test sample measurement solution: precisely weighing 500mg of cefprozil test sample, placing the cefprozil test sample in a 10ml volumetric flask, adding 10% sodium bicarbonate solution, ultrasonically dissolving, diluting to a scale, and shaking uniformly to obtain test sample solution; taking 10ml Coomassie brilliant blue G-250 dye solution, placing in a penicillin bottle, adding 200 μ L of test solution, sealing, mixing, standing for 10min, and using as test solution.
Placing the test solution into an ultraviolet spectrophotometer, measuring the light absorption value at 595nm, calculating the concentration of the protein contained in the test solution according to a standard curve method, and further converting the enzyme residue content in the cefprozil sample prepared by the enzyme method, wherein the result is shown in the following table 4:
table 4: calculation of residual enzyme content in Cefprozil sample
| Batch number | Absorbance value | Residual protein concentration (μ g/mL) | Residual enzyme protein% |
| 1 | 0.016 | ＜LOQ | ＜LOQ |
| 2 | 0.023 | ＜LOQ | ＜LOQ |
| 3 | 0.020 | ＜LOQ | ＜LOQ |
| 4 | 0.032 | 13.94 | 0.028％ |
| 5 | 0.043 | 22.54 | 0.045％ |
4. In this example, the accuracy, reproducibility and reliability of the method for detecting the enzyme protein residue in cefprozil prepared by the enzymatic method of the present invention are proved by methodology verification.
4.1 precision
(1) Analysis of reproducibility
Repeatedly establishing a standard curve according to the standard curve establishing method; taking a test sample (batch number is 5), and determining the protein content of the test sample according to the method for determining the protein content in the test sample determination solution; the results of the standard curve measurements are shown in table 5 and fig. 2: the test results of the test samples are shown in Table 6;
table 5: standard curve test result for analysis of repeatability
| Relative to the concentration level (g/g) of the sample | Protein concentration (. mu.g/mL), x | Absorbance value, y |
| 0.05％ | 24.305 | 0.047 |
| 0.2％ | 97.221 | 0.074 |
| 0.4％ | 194.442 | 0.115 |
| 0.8％ | 388.885 | 0.249 |
| 1.2％ | 583.327 | 0.354 |
| 1.6％ | 777.769 | 0.458 |
| 2.0％ | 972.211 | 0.549 |
The linear regression equation from fig. 2 is: y 0.000548654x + 0.0255115; r is2＝0.99696。
Table 6: test result of test article (lot number 5)
| Batch number | Absorbance value | Residual protein concentration (μ g/mL) | Residual enzyme protein% |
| 5 | 0.038 | 22.76 | 0.045 |
Taking the same sample (batch number is 5) to prepare 6 parts of sample solution in parallel; respectively taking 10ml of Coomassie brilliant blue G-250 dye solution, respectively placing the Coomassie brilliant blue G-250 dye solution into 6 penicillin bottles, respectively adding 100 mu L of linear solution which is equivalent to 0.2% of a test sample in a standard curve and 100 mu L of the test sample solution, uniformly sealing and mixing, and standing for 10min to obtain 6 analysis repeatability solutions; putting the 6 analysis repetitive solutions into an ultraviolet spectrophotometer, reading an absorbance value at 595nm, and calculating by a standard curve to obtain the concentration of protein contained in the analysis repetitive solutions; and subtracting the protein concentration of the corresponding test solution from the protein concentration of the analytical repetitive solution to obtain the protein concentration of the actually added linear solution, wherein the ratio of the actually added protein concentration to the theoretically added protein concentration is the recovery rate. RSD was calculated for the reproducibility recovery of each analysis and the results are shown in table 7 below.
Table 7: analysis of the results of repeated measurements
As a result, the RSD of 6 analysis repetitive solutions is 4.6%, and the detection method for the enzyme protein residue in the cefprozil prepared by the enzyme method has good repeatability.
(2) Intermediate precision
Establishing a standard curve according to the standard curve establishing method on different dates, taking a sample (batch number is 5), and determining the protein content of the sample according to the method in the embodiment; the standard curve detection result is shown in 8 and figure 3: the test results of the test samples are shown in Table 9.
Table 8: test result of standard curve made for intermediate precision
| Relative to the concentration level (g/g) of the sample | Protein concentration (. mu.s)g/mL)，x | Absorbance value, y |
| 0.05％ | 24.305 | 0.016 |
| 0.2％ | 97.221 | 0.060 |
| 0.4％ | 194.442 | 0.123 |
| 0.8％ | 388.885 | 0.263 |
| 1.2％ | 583.327 | 0.387 |
| 1.6％ | 777.769 | 0.477 |
| 2.0％ | 972.211 | 0.626 |
The linear regression equation from FIG. 3 is: y 0.000636480x + 0.00257969; r is2＝0.99730。
Table 9: test result of test article (lot number 5)
| Batch number | Absorbance value | Residual protein concentration (μ g/mL) | Residual enzyme protein% |
| 5 | 0.017 | 22.66 | 0.045 |
6 portions of intermediate precision solutions were prepared and the protein concentration was determined in the same operation as for analytical reproducibility. The RSD of the actual recovery rate and the recovery rate of each intermediate precision solution were calculated, and the results are shown in table 10 below;
TABLE 10 results of intermediate precision measurements
As a result, the RSD of 6 parts of the intermediate precision solution was 3.0%, and the reproducibility of the detection method was good on different dates. Comparing the analytical reproducibility with the RSD of the recovery rate of the sample-adding solution with intermediate precision, the results are shown in Table 11;
table 11: results of precision measurement
As a result, the recovery rate RSD of 12 parts of data detected on different dates is 4.5%, and the detection method for detecting the enzyme protein residue in the cefprozil prepared by the enzyme method in the embodiment has good precision.
(3) Accuracy of
According to the above-mentioned markThe standard curve is established firstly, and the linear regression equation of the standard curve is obtained as follows: y 0.000548654x + 0.0255115; r is2＝0.99696。
Taking a test sample (batch number is 5), preparing a test sample solution, measuring the protein content of the test sample solution according to the method of the embodiment, and measuring the detection result to be 22.76 mu g/mL;
preparation of accuracy solution 1: taking 10ml of Coomassie brilliant blue G-250 dye solution, placing the Coomassie brilliant blue G-250 dye solution in a penicillin bottle, adding 100 mu L of linear solution which is equivalent to 0.05% of a test sample in a standard curve and 100 mu L of the test sample solution, sealing, uniformly mixing, and standing for 10min to obtain an accuracy solution 1; preparing 3 parts in parallel;
preparation of accuracy solution 2: taking 10ml of Coomassie brilliant blue G-250 dye solution, placing the Coomassie brilliant blue G-250 dye solution in a penicillin bottle, adding 100 mu L of linear solution which is equivalent to 0.2% of a test sample in a standard curve and 100 mu L of the test sample solution, sealing, uniformly mixing, and standing for 10min to obtain an accuracy solution 2; preparing 3 parts in parallel;
preparation of accuracy solution 3: taking 10ml of Coomassie brilliant blue G-250 dye solution, placing the Coomassie brilliant blue G-250 dye solution in a penicillin bottle, adding 100 mu L of linear solution which is 1.2 percent of a test sample in a standard curve and 100 mu L of the test sample solution, sealing, uniformly mixing, and standing for 10min to obtain an accuracy solution 3; preparing 3 parts in parallel;
accuracy solution 4 preparation: taking 10ml of Coomassie brilliant blue G-250 dye solution, placing the Coomassie brilliant blue G-250 dye solution in a penicillin bottle, adding 100 mu L of linear solution which is equivalent to 2.0% of a test sample in a standard curve and 100 mu L of the test sample solution, sealing, uniformly mixing, and standing for 10min to obtain an accuracy solution 4; preparing 3 parts in parallel;
placing the 12 parts of the accuracy solution into an ultraviolet spectrophotometer, reading an absorbance value at 595nm, and calculating by a standard curve to obtain the concentration of protein contained in the analysis repetitive solution; and subtracting the protein concentration of the test solution from the protein concentration of the accuracy solution to obtain the protein concentration of the actually added linear solution, wherein the ratio of the actually added protein concentration to the theoretically added protein concentration is the recovery rate. RSD was calculated for solution recovery for each accuracy, and the results are shown in table 12 below.
Table 12: results for recovery of 12 solutions of accuracy
As a result, the method for detecting the enzyme protein residue in cefprozil prepared by the enzyme method has good accuracy.
As can be seen from the concentrations of the linear solutions of the standard curve, the protein solution with the concentration of 24.305 mu g/ml to 972.211 mu g/ml is considered, and is approximately equal to 0.05 percent to 2.0 percent of the concentration of the test sample; the accuracy also inspects the concentration solution of 22.75-498.46 mug/ml, which is equivalent to 0.05-1.0% of the concentration of the test sample; the method for detecting the residual enzyme protein in the cefprozil prepared by the enzyme method has the advantages that the concentration of the residual enzyme protein in the cefprozil sample prepared by the enzyme method is about 0.05-1.0 percent by weight, the repeatability of the detection result is better, and the accuracy is higher.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. 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.
Claims (10)
1. A detection method for enzyme protein residue in cefprozil prepared by an enzyme method is characterized by comprising the following steps:
preparing Coomassie brilliant blue G-250 dye solution and standard protein stock solution;
preparation of standard curve solution: taking standard protein stock solutions, and diluting with sodium bicarbonate solutions with the same concentration and different volumes respectively to obtain respective standard protein linear solutions; respectively adding the Coomassie brilliant blue G-250 dye solution with the same volume into the standard protein linear solutions, and uniformly mixing; standing for reaction to obtain a standard curve solution;
preparation of a blank solution: taking Coomassie brilliant blue G-250 dye solution with the same volume as that of the standard curve solution during preparation, adding sodium bicarbonate solution with the same concentration, mixing uniformly, standing for reaction, and taking the mixture as blank solution;
preparation of test article assay solution: weighing a proper amount of cefprozil test sample, adding sodium bicarbonate solution with the same concentration for dissolving and diluting to obtain test sample solution; adding the Coomassie brilliant blue G-250 dye solution with the same volume as that of the standard curve solution during preparation into the test solution, mixing uniformly, and standing for reaction to obtain a test solution;
using an ultraviolet spectrophotometer, correcting through a blank solution, determining absorbance by adopting a standard curve solution, and drawing a standard curve according to the concentration and absorbance value of each protein linear solution in the standard curve solution; and calculating the protein concentration in the test solution of the test sample by adopting a standard curve method according to the absorbance value read by the test solution of the test sample, thereby calculating the residual content of the enzyme protein contained in the test sample.
2. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 1, which is characterized in that: further comprising the steps of:
preparation of Coomassie brilliant blue G-250 dye solution: weighing Coomassie brilliant blue G-250, dissolving in ethanol, adding appropriate amount of phosphoric acid, and diluting to constant volume with purified water;
preparation of standard protein stock solution: weighing a proper amount of bovine serum albumin, and dissolving and diluting the bovine serum albumin with purified water to obtain a standard protein stock solution.
3. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 2, which is characterized in that: in the preparation process of the Coomassie brilliant blue G-250 dye solution, the ratio of the weight of the Coomassie brilliant blue G-250 to the volume of ethanol to the volume of phosphoric acid to the volume of constant volume is 5G: (2-3) L: (5-6) L: 50L.
4. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 1, which is characterized in that: the concentration of the standard protein stock solution is 0.1-5 mg/ml, and the concentration of the test solution is 5-100 mg/ml.
5. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 1, which is characterized in that: the concentration of the sodium bicarbonate solution is 1-15% g/ml.
6. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 1, which is characterized in that: the standing reaction time during the preparation of the blank solution and the standing reaction time during the preparation of the standard curve solution are the same as the standing reaction time during the preparation of the test sample determination solution; the volume of the standard protein linear solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the standard curve solution, the volume of the test sample solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the test sample determination solution and the volume of the sodium bicarbonate solution added during the preparation of the blank solution are equal.
7. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 1, which is characterized in that: the standing reaction time during the preparation of the blank solution, the standing reaction time during the preparation of the standard curve solution and the standing reaction time during the preparation of the test sample determination solution are all 2-20 minutes.
8. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 1, which is characterized in that: the wavelength range is 590-600 nm when the ultraviolet spectrophotometer is used.
9. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 1, which is characterized in that: at least five standard protein linear solutions were prepared.
10. The method for detecting enzyme protein residues in cefprozil prepared by the enzyme method according to claim 1, which is characterized in that:
the preparation method of the Coomassie brilliant blue G-250 dye solution comprises the following steps: weighing 100mg of Coomassie brilliant blue G-250, dissolving in 40-60 ml of ethanol, adding 120ml of phosphoric acid 100 and adding purified water to a constant volume of 1000 ml;
the standard protein stock solution has the concentration of 0.1-5 mg/ml, and the standard protein linear solution has the following concentrations: 25 mu g/ml, 100 mu g/ml, 200 mu g/ml, 400 mu g/ml, 600 mu g/ml, 800 mu g/ml and 1000 mu g/ml, wherein the concentration c of the sodium bicarbonate solution is 1-15% (g/ml);
the concentration of the test solution is 5-100 mg/ml;
the standing reaction time during the preparation of the blank solution, the standing reaction time during the preparation of the standard curve solution and the standing reaction time during the preparation of the test sample determination solution are all 2-20 minutes, the volumes of the Coomassie brilliant blue G-250 dye solutions adopted by the standard curve solution, the blank solution and the test sample determination solution are all 5-25 ml, and the volumes of the standard protein linear solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the standard curve solution, the test sample solution added into the Coomassie brilliant blue G-250 dye solution during the preparation of the test sample determination solution and the sodium bicarbonate solution added during the preparation of the blank solution are all 10-250 muL.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112147095A (en) * | 2020-09-22 | 2020-12-29 | 上海昊海生物科技股份有限公司 | Method for rapidly determining crosslinked sodium hyaluronate gel protein residue |
| CN112945883A (en) * | 2021-03-30 | 2021-06-11 | 南京林业大学 | Enzyme residue detection method for preparing medical intermediate by enzyme method |
| CN117451650A (en) * | 2023-12-25 | 2024-01-26 | 山东齐都药业有限公司 | Detection method for determining protein residual quantity in levocarnitine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4023933A (en) * | 1976-06-10 | 1977-05-17 | The University Of Georgia | Protein-assay reagent and method |
| WO2004110399A2 (en) * | 2003-06-19 | 2004-12-23 | Ranbaxy Laboratories Limited | Solvates of cefprozil |
| CN101566575A (en) * | 2009-04-29 | 2009-10-28 | 东北制药总厂 | Method for detecting protein content in 2-keto-L-gulonic acid |
| CN102507466A (en) * | 2011-10-20 | 2012-06-20 | 济南大学 | Improved spectrophotometry method for determining proteins by using Coomassie brilliant blue |
| CN103674942A (en) * | 2013-11-25 | 2014-03-26 | 北京工业大学 | Method for measuring protein in sludge according to coomassie brilliant blue method |
-
2019
- 2019-12-26 CN CN201911368630.3A patent/CN110954392A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4023933A (en) * | 1976-06-10 | 1977-05-17 | The University Of Georgia | Protein-assay reagent and method |
| WO2004110399A2 (en) * | 2003-06-19 | 2004-12-23 | Ranbaxy Laboratories Limited | Solvates of cefprozil |
| CN101566575A (en) * | 2009-04-29 | 2009-10-28 | 东北制药总厂 | Method for detecting protein content in 2-keto-L-gulonic acid |
| CN102507466A (en) * | 2011-10-20 | 2012-06-20 | 济南大学 | Improved spectrophotometry method for determining proteins by using Coomassie brilliant blue |
| CN103674942A (en) * | 2013-11-25 | 2014-03-26 | 北京工业大学 | Method for measuring protein in sludge according to coomassie brilliant blue method |
Non-Patent Citations (1)
| Title |
|---|
| 杜琳等: "酶法合成熊去氧胆酸中蛋白质含量的测定方法比较研究", 《成都师范学院学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112147095A (en) * | 2020-09-22 | 2020-12-29 | 上海昊海生物科技股份有限公司 | Method for rapidly determining crosslinked sodium hyaluronate gel protein residue |
| CN112945883A (en) * | 2021-03-30 | 2021-06-11 | 南京林业大学 | Enzyme residue detection method for preparing medical intermediate by enzyme method |
| CN117451650A (en) * | 2023-12-25 | 2024-01-26 | 山东齐都药业有限公司 | Detection method for determining protein residual quantity in levocarnitine |
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