CN115508464A - Analysis method for activity of cefradine synthetase - Google Patents
Analysis method for activity of cefradine synthetase Download PDFInfo
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Abstract
The invention discloses an analysis method of activity of cefradine synthetase, belonging to the field of chemical analysis, and the activity of the synthetase is obtained by calculation after the content of a reaction solution to be detected of cefradine is determined by adopting a high performance liquid chromatography, and the method comprises the following steps: preparing a reference substance solution, preparing a reaction solution of a sample to be detected, and analyzing the activity of the synthetase by high performance liquid chromatography. The operation process is simple and convenient, the accuracy is high, the stability is good, the durability is excellent, and the influence of impurities on the measurement result can be effectively avoided; the method has the advantages of low requirement on equipment, simple and easily-obtained mobile phase medium and strong feasibility and applicability.
Description
Technical Field
The invention relates to an analysis method for activity of cefradine synthetase, belonging to the field of chemical analysis.
Background
Cephradine is a first generation cephalosporin having the following structural formula:
at present, the enzymatic method of cefradine in domestic enterprises is still in the research and development stage, no suitable detection method exists for immobilized penicillin acylase used for synthesizing cefradine, and a detection method needs to be developed for effectively evaluating the synthesis activity of the immobilized penicillin acylase.
Disclosure of Invention
In view of the above, the present invention provides a method for analyzing the activity of cephradine synthetase, so as to solve the problem that the synthesis activity of immobilized penicillin acylase for synthesizing cephradine cannot be effectively evaluated in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for analyzing activity of cefradine synthetase comprises the following steps of measuring the content of a reaction solution to be measured of cefradine by high performance liquid chromatography, and obtaining the activity of the synthetase by calculation:
(1) Preparation of control solutions: precisely weighing a cefradine reference substance, adding a mobile phase for dissolving, quantitatively diluting to prepare a cefradine reference substance solution with the concentration of 0.05-1.0 mg/mL, and shaking up;
(2) Preparation of a sample solution to be tested:
preparation of a substrate solution: weighing 4.3g of 7-ADCA and 4.1g of dihydroglycine methyl ester hydrochloride, adding 200ml of purified water for dissolving, and adjusting the pH value to be =7.0 by using ammonia water for later use;
preparing cefradine synthetase: taking a proper amount of cefradine enzyme, washing with 100ml of purified water, carrying out suction filtration until no water drops, and precisely weighing 2.0g for later use;
preparing a reaction solution sample: starting a water area constant-temperature oscillating screen, setting the temperature to be 20 ℃, oscillating the water area constant-temperature oscillating screen at the oscillation frequency of 265, accurately measuring 90ml of substrate solution, placing the substrate solution in a 250 conical flask, oscillating the substrate solution for 10min to ensure that the temperature of the solution is 20 ℃, adding 2.0g of cefradine synthetase, washing the bottleneck with 10ml of purified water, timing for 10min, and keeping the timing for later use;
preparing a solution of a sample to be detected: precisely measuring a reaction solution sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a to-be-measured sample solution with the concentration of 0.05-1.0 mg/mL, and shaking up;
(3) Analysis of synthetase Activity: respectively and precisely measuring 10 mu L of each of the solution of the sample to be measured and the solution of the reference substance, injecting the solution of the sample to be measured and the solution of the reference substance into a high performance liquid chromatograph, recording chromatograms of the sample to be measured and the reference substance, and calculating by peak area according to an external standard method to obtain the concentration of cefradine in the sample to be measured, thereby calculating the activity of the synthetase, wherein the calculation formula is as follows:
enzyme activity calculation formula:
wherein f is a correction factor; a is the concentration (mmol/L) of cefradine in the reaction solution, t is the reaction time (min), m is the mass (g) of the enzyme sample, the molecular weight of cefradine is 349.41 (without water), and U is the activity (mmol/min) of the enzyme.
On the basis of the technical scheme, the invention can be further improved as follows:
further, the chromatographic conditions were: octadecylsilane chemically bonded silica is used as a filling agent, the flow rate of a mobile phase is 0.8-1.2 mL/min, the detection wavelength is 249-259 nmnm, the column temperature is 20-30 ℃, and the number of theoretical plates is not less than 2000 calculated according to a main peak.
Further, the mobile phase is a mixed solution of water, methanol, a 3.86% sodium acetate solution and a 4% acetic acid solution, the volume ratio of the water, the methanol, the 3.86% sodium acetate solution and the 4% acetic acid solution is 3710:1200: 70-80: 14-16, and the following ratio of the volume ratio of the water, the methanol, the 3.86% sodium acetate solution and the 4% acetic acid solution is 3710.
Further, the preparation method of the 3.86% sodium acetate solution comprises the following steps: weighing 64.0g of sodium acetate trihydrate, dissolving in 500ml of ultrapure water, and adding ultrapure water to dilute to 1000ml after dissolving;
the preparation method of the 4% acetic acid solution comprises the following steps: measuring 9.6ml of glacial acetic acid, dissolving in 250ml of ultrapure water, and mixing uniformly.
Further, the flow rate of the mobile phase is 1.0mL/min;
the column temperature is 25 ℃;
the detection wavelength is 254nm.
Further, the concentration of the reference substance solution and the tested sample solution in the step (3) is 0.5mg/mL.
The method for analyzing the activity of the cefradine synthetase by the high performance liquid chromatography has the advantages of simple and convenient operation process, high accuracy, good stability and excellent durability, and can effectively avoid the influence of impurities on the measurement result; the method has low requirement on equipment, simple and easily obtained mobile phase medium and strong feasibility and applicability.
Drawings
FIG. 1 is a chromatogram of a cefradine standard of example 2 of the present invention;
FIG. 2 is a chromatogram of a sample of a reaction solution of cefradine synthase in example 2 of the present invention;
FIG. 3 is a standard curve diagram of the linear assay for determining the content of cefradine in example 2 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
In the following embodiments, the invention employs the following instruments and reagents:
shimadzu high performance liquid chromatography (Shimadzu SPD-10A detector, shimadzu LC-10AT pump);
sample and reagent: methanol (chromatographic purity) and a cefradine reference substance (batch number: CU211201, identification amount: 93.6%).
The method for measuring the content of the cefradine in the reaction liquid of the tested sample of the activity of the cefradine synthetase by adopting the high performance liquid chromatography so as to calculate the activity of the synthetase comprises the following steps:
(a) Preparation of control solutions: precisely weighing a cefradine reference substance, adding a mobile phase for dissolving, quantitatively diluting to prepare a cefradine reference substance solution with the concentration of 0.05-1.0 mg/mL, shaking up, and fixing the volume;
(b) Preparation of a tested sample solution: preparation of substrate solution: weighing 4.3g of 7-ADCA and 4.1g of dihydrophenyl glycine methyl ester hydrochloride, adding 200ml of purified water for dissolving, and adjusting the pH value to be =7.0 by using ammonia water for later use; cephradine synthase: taking a proper amount of cefradine enzyme, washing with 100ml of purified water, carrying out suction filtration until no water drops, and precisely weighing 2.0g for later use; starting a water area constant-temperature oscillating screen, setting the temperature to be 20 ℃, oscillating at the frequency of 265, accurately measuring 90ml of substrate solution, placing the substrate solution into a 250 conical flask, oscillating for 10min to enable the solution temperature to be 20 ℃, adding 2.0g of enzyme, washing the flask opening with 10ml of purified water, timing for 10min, and ending timing; precisely measuring a reaction solution sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a to-be-measured sample solution with the concentration of 0.05-1.0 mg/mL, shaking up, and fixing the volume;
(c) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, a mixed solution of water, methanol, a 3.86% sodium acetate solution and a 4% acetic acid solution in a volume ratio of 3710; the detection wavelength is 254nm; the column temperature was 25 ℃; the theoretical plate number is not lower than 2000 calculated according to the main peak;
(d) The determination method comprises the following steps:
weighing a proper amount of cefradine reference substance according to the 2015 edition of Chinese pharmacopoeia, precisely weighing, adding a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution in a volume ratio of 3710;
taking a proper amount of reaction liquid of the test sample, precisely measuring, measuring by the same method, and recording a chromatogram;
calculating the concentration of the cefradine in the sample to be detected by the peak area according to an external standard method, thereby calculating the activity of the synthetase:
enzyme activity calculation formula:
wherein f is a correction factor; a is the concentration (mmol/L) of cefradine in the reaction solution, t is the reaction time (min), m is the mass (g) of the enzyme sample, the molecular weight of cefradine is 349.41 (without water), and U is the activity (mmol/min) of the enzyme.
Example 1
Chromatographic conditions are as follows: by using a Dima C 18 Reversed phase column 4.6mm × 250mm,5 μm; the mobile phase is a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution with the volume ratio of 3710; the flow rates are respectively 0.8mL/min, 1.0mL/min and 1.2mL/min; an ultraviolet detector with the detection wavelength of 254nm; the column temperature was 25 deg.C (room temperature); the theoretical plate number is not lower than 2000 calculated by main peak.
The measured sample solution: preparation of substrate solution: weighing 4.3g of 7-ADCA and 4.1g of dihydrobenzene glycine methyl ester hydrochloride, adding 200ml of purified water for dissolving, and adjusting the pH value to be =7.0 by using ammonia water for later use; cephradine synthase: taking a proper amount of cefradine enzyme, washing with 100ml of purified water, carrying out suction filtration until no water drops flow out, and precisely weighing 2.0g for later use; starting a water area constant-temperature oscillating screen, setting the temperature to be 20 ℃, oscillating at the frequency of 265, accurately measuring 90ml of substrate solution, placing the substrate solution into a 250 conical flask, oscillating for 10min to enable the solution temperature to be 20 ℃, adding 2.0g of enzyme, washing the flask opening with 10ml of purified water, timing for 10min, and ending timing; precisely measuring a reaction solution sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a measured sample solution with the concentration of 0.05mg/mL, shaking up, and fixing the volume; control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain 0.02mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the determination method comprises the following steps: precisely measuring 10 μ L of the sample solution to be measured and the cefradine reference solution respectively, and injecting into a liquid chromatograph; and recording chromatograms of the reference substance and the detected sample, and calculating according to an external standard method by using peak areas to obtain a determination result of the content of the cefradine in the sample.
Calculating the content of cefradine by adopting the following steps so as to calculate the activity of the synthetase:
enzyme activity calculation formula:
wherein f is a correction factor; a is the concentration (mmol/L) of the cefradine in the reaction solution, t is the reaction time (min), m is the mass (g) of the enzyme sample, the molecular weight of the cefradine is 349.41 (without water), and U is the activity (mmol/min) of the enzyme.
The concentrations of the cefradine in the sample obtained by the calculation by the method are 53.51, 52.98 and 53.24, and the enzyme activities are 266.84, 264.20 and 265.50.
Example 2
The chromatographic conditions were the same as in example 1.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.5mg of cefradine sample in each 1mL of solution, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.5mg of cefradine as reference substance per 1mL, shaking, and fixing volume;
the determination method and the steps for calculating the concentration of cefradine are the same as in example 1, the chromatograms of cefradine reference substance and cefradine sample are shown in fig. 1-3, the concentration of cefradine in the obtained sample is calculated to be 53.59, 53.57 and 53.61, and the enzyme activity is calculated to be 267.28, 266.20 and 267.39.
Example 3
The chromatographic conditions were the same as in example 1.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine sample in each 1mL of solution, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as a reference substance, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine as the reference substance in every 1mL, shaking up, and fixing the volume;
the procedure for determining the assay and calculating the concentration of cephradine is the same as in example 1, the concentrations of cephradine in the samples are calculated to be 53.92, 54.01 and 53.73, respectively, and the enzyme activities are calculated to be 268.89, 269.34 and 267.94.
Example 4
The chromatographic conditions were similar to example 1, except that the mobile phase used was a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution in a volume ratio of 3710.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.05mg of cefradine sample in each 1mL of cefradine sample, shaking uniformly, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase pyran for dissolution, quantitatively diluting to obtain a solution containing 0.05mg of cefradine reference substance per 1mL, shaking up, and fixing volume;
the steps of measuring and calculating the concentration of cefradine are the same as in example 1, the concentration of cefradine in the sample is calculated to be 53.51, 53.22 and 53.29 respectively, and the enzyme activity is calculated to be 266.84, 265.4 and 265.75.
Example 5
The chromatographic conditions were the same as in example 4.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.5mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.5mg of cefradine as reference substance per 1mL, shaking, and fixing volume;
the steps of the determination method and the calculation of the concentration of cefradine are the same as those in example 1, the concentrations of cefradine in the obtained samples are calculated to be 53.77, 53.47 and 53.52 respectively, and the enzyme activities are calculated to be 268.14, 266.64 and 266.89.
Example 6
The chromatographic conditions were the same as in example 4.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain a solution containing 1.0mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the steps of the determination and calculation of the concentration of cephradine are the same as in example 1, the concentrations of cephradine in the sample are calculated to be 53.92, 53.97 and 53.73, respectively, and the enzyme activities are calculated to be 268.89, 269.14 and 267.94.
Example 7
The chromatographic conditions were similar to example 1, except that the mobile phase used was a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution in a volume ratio of 3710.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.05mg of the cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.05mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the steps of the determination method and the calculation of the concentration of cefradine are the same as in example 1, the concentrations of cefradine in the sample obtained by calculation are respectively 53.28, 53.43 and 53.52, and the enzyme activities are calculated to be 265.70, 266.44 and 266.94.
Example 8
The chromatographic conditions were the same as in example 7.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.5mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.5mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the steps of the determination and calculation of the concentration of cephradine are the same as in example 1, the concentrations of cephradine in the sample are calculated to be 53.59, 53.27 and 53.82, respectively, and the enzyme activities are calculated to be 267.28, 265.65 and 268.39.
Example 9
The chromatographic conditions were the same as in example 7.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain a solution containing 1.0mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the steps of measuring and calculating the concentration of cefradine are the same as in example 1, the concentration of cefradine in the sample is calculated to be 53.27, 53.96 and 53.80 respectively, and the enzyme activity is calculated to be 265.65, 269.07 and 268.49.
Example 10
The chromatographic conditions were similar to those of example 1, except that the mobile phase used was a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution in a volume ratio of 3710.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.05mg of the cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.05mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the procedure for determining the assay and calculating the concentration of cephradine is the same as in example 1, and the concentrations of cephradine in the samples are calculated as follows: 53.27, 54.23 and 53.93, and calculating to obtain the enzyme activities 265.65, 270.44 and 268.94.
Example 11
The chromatographic conditions were the same as in example 10.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.5mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.5mg of cefradine as reference substance per 1mL, shaking, and fixing volume;
the procedure for determining the assay and calculating the concentration of cephradine is the same as in example 1, and the concentrations of cephradine in the samples are calculated as follows: 53.26, 54.19 and 53.83, and the enzyme activity is calculated to be 265.65, 270.29 and 269.49.
Example 12
The chromatographic conditions were the same as in example 10.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 1.0g of cefradine reference substance per 1mL, shaking, and fixing volume;
the procedure for determining the assay and calculating the concentration of cephradine is the same as in example 1, the concentrations of cephradine in the samples are calculated to be 53.57, 53.89 and 54.12, respectively, and the enzyme activities are calculated to be 267.15, 268.74 and 269.89.
Example 13
The chromatographic conditions were similar to example 1, except that the mobile phase used was a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution in a volume ratio of 3710.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.05mg of the cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.05mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the steps of the determination method and the calculation of the concentration of cefradine are the same as in example 1, the concentrations of cefradine in the sample obtained by calculation are respectively 53.26, 54.29 and 53.83, and the enzyme activities are calculated to be 265.67, 278.29 and 269.99.
Example 14
The chromatographic conditions were the same as in example 13.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.5mg of cefradine sample in each 1mL of solution, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.5mg of cefradine as reference substance per 1mL, shaking, and fixing volume;
the steps of the determination method and the calculation of the content of the cefradine are the same as the step of the example 1, the content of the cefradine in the sample obtained by calculation is respectively 53.26, 53.99 and 53.84, and the enzyme activity is calculated to be 265.65, 270.29 and 279.49.
Example 15
The chromatographic conditions were the same as in example 13.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine sample in each 1mL of solution, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain a solution containing 1.0mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the steps of the determination method and the calculation of the content of the cefradine are the same as the step of the example 1, the content of the cefradine in the sample obtained by calculation is respectively 53.36, 53.67 and 54.13, and the enzyme activity is calculated to be 270.65, 272.29 and 279.49.
Example 16
The chromatographic conditions were similar to example 1, except that the mobile phase used was a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution in a volume ratio of 3710.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.05mg of the cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.05mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the steps of the determination method and the calculation of the content of the cefradine are the same as the steps in the example 1, the content of the cefradine in the sample obtained by calculation is respectively 54.26, 54.19 and 53.83, and the enzyme activity is calculated to be 275.85, 270.29 and 267.46.
Example 17
The chromatographic conditions were the same as in example 16.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.5mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine reference substance, adding mobile phase for dissolving, quantitatively diluting to obtain solution containing 0.5mg of cefradine reference substance per 1mL, shaking, and fixing volume;
the steps of the determination method and the calculation of the content of the cephradine are the same as the step of the example 1, the content of the cephradine in the sample obtained by calculation is respectively 53.26, 54.36 and 53.83, and the enzyme activity is calculated to be 265.68, 270.26 and 269.88.
Example 18
The chromatographic conditions were the same as in example 16.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as a reference substance, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine as the reference substance in every 1mL, shaking up, and fixing the volume;
the steps of the determination method and the calculation of the content of the cephradine are the same as the step of the example 1, the content of the cephradine in the sample obtained by calculation is respectively 53.27, 54.19 and 53.93, and the enzyme activity is calculated to be 265.95, 270.29 and 268.99.
Example 19
The chromatographic conditions were similar to those of example 1, except that the mobile phase used was a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution in a volume ratio of 3710.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.02mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as a reference substance, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.02mg of cefradine as the reference substance in every 1mL, shaking up, and fixing the volume;
the steps of the determination method and the calculation of the content of the cefradine are the same as the steps in the example 1, the content of the cefradine in the sample obtained by calculation is respectively 53.29, 54.19 and 53.93, and the enzyme activity is calculated to be 265.98, 271.29 and 269.79.
Example 20
The chromatographic conditions were the same as in example 19.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.2mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as a reference substance, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 0.2mg of cefradine as the reference substance in every 1mL, shaking up, and fixing the volume;
the steps of the determination method and the calculation of the content of the cefradine are the same as the steps in the example 1, the content of the cefradine in the sample obtained by calculation is respectively 53.26, 54.19 and 53.83, and the enzyme activity is calculated to be 265.65, 270.29 and 269.49.
Example 21
The chromatographic conditions were the same as in example 19.
The measured sample solution: precisely weighing a cefradine sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine sample in every 1mL, shaking up, and fixing the volume;
control solution: precisely weighing cefradine as a reference substance, adding a mobile phase for dissolving, quantitatively diluting to prepare a solution containing 1.0mg of cefradine as the reference substance in every 1mL, shaking up, and fixing the volume;
the steps of the determination method and the calculation of the content of the cefradine are the same as the steps in the example 1, the content of the cefradine in the sample obtained by calculation is respectively 53.26, 54.19 and 53.83, and the enzyme activity is calculated to be 265.65, 270.29 and 269.49.
From the above examples, it can be seen that: example 2 is a preferred protocol for analyzing the content of cephradine.
The following methodological validation was performed for the method of example 2:
example 22
System suitability test
Chromatographic conditions are as follows: mobile phase: water: methanol: 3.86% sodium acetate solution: 4% acetic acid solution =3710, wavelength: 254nm, flow rate: 1.0ml/min. The column temperature is 25 +/-1 ℃; and (3) chromatographic column: dima C18250mm multiplied by 4.6mm 5.0um; the theoretical plate number is not lower than 2000 calculated according to the main peak; (3.86% sodium acetate solution: weighing 64.0g sodium acetate trihydrate and dissolving in 500ml ultrapure water, adding ultrapure water and diluting to 1000ml 4% acetic acid solution: weighing 9.6ml glacial acetic acid and dissolving in 250ml ultrapure water).
Control solution: precisely weighing 25mg of cefradine reference substance, placing the cefradine reference substance in a 50ml volumetric flask, diluting the cefradine reference substance with a mobile phase to a constant volume to a scale, wherein the concentration is about 0.5mg/ml.
Test solution: precisely measuring 1ml of the feed liquid, putting the feed liquid into a 50ml volumetric flask, diluting the feed liquid with a mobile phase to a constant volume to a scale, wherein the concentration is about 0.5mg/ml.
And (3) testing: taking 10 mu L of cefradine control solution, and injecting into a liquid chromatograph; measuring 6 times by the same method, and recording chromatogram of the control. The results of the experiment are shown in table 1.
TABLE 1 table of the results of the test of the applicability of the cefradine content measuring system
As can be seen from Table 1, the relative standard deviation RSD calculated 6 times according to the peak area of the sample is 0.25%, which meets the requirement that RSD is less than or equal to 1.0%; the theoretical plate number is 9987 calculated according to the main peak, and the requirement of not less than 2000 is met. The test result shows that the system repeatability of the method is good.
Example 23
Method precision test
6 parts of test solution are prepared according to the method of example 22, and the same chromatographic conditions as the previous method are adopted, the sample injection measurement is carried out according to the measurement method of example 22, the atlas is recorded, and the content of the cephradine in each test solution is calculated according to the external standard method, and the result is shown in Table 2.
TABLE 2 measurement of cefradine content precision test results
RSD was calculated to be 0.04% for 6 data, less than limit 1.0%. The test result shows that the method has good precision.
Example 24
Intermediate precision test
6 parts of test solution was prepared according to the method of example 22, and tested by different analysts on different dates using different instruments according to the method of example 22, chromatograms were recorded and analyzed by summarizing 3 sets of data, relative standard deviations of the results were calculated as shown in Table 3, and the data in the intermediate precision test were compared with six data in the method precision test.
TABLE 3 intermediate precision test results of cefradine concentration determination
RSD of 6 data in intermediate precision was calculated to be 0.04%, less than limit 1.0%. The test result shows that the method has good intermediate precision.
Example 25
Investigation of linear relationships
A control solution was prepared according to the method of example 22 and measured according to the same chromatographic conditions as in example 22: injecting 10 mu L of the left cefradine solution into a liquid chromatograph; measuring 6 times by the same method, and recording reference chromatogram as system applicability experiment.
Taking 100mg of a cefradine reference substance, placing the cefradine reference substance in a 100mL volumetric flask, adding a mobile phase solution to dilute the cefradine into a storage solution with the cefradine of 1mg/mL, precisely measuring 1mL, 3mL, 5mL, 7mL and 10mL of the reference storage solution, placing the reference storage solution in a 10mL volumetric flask, diluting the solution to a scale by using the mobile phase, shaking the solution uniformly to prepare solutions containing 0.1mg, 0.3mg, 0.5mg, 0.7mg and 1mg of cefradine in each 1mL of the solution (corresponding to 20%, 60%, 100%, 140% and 200% of a test solution), taking 10 mu L of the solutions as linear solutions, injecting the linear solutions into a liquid chromatograph, respectively recording chromatograms, drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and calculating a regression equation y =24467 x-216.2 as shown in a figure 3, wherein the corresponding data is shown in a table 4; the correlation coefficient R is 0.9998, and meets the requirement that the correlation coefficient R of the components to be tested in the linear range test is more than or equal to 0.999. The test result shows that: the cefradine has a good linear relation between the peak area and the concentration within the concentration range of 0.1-1.0 mg/mL.
TABLE 4 Linear test results for cefradine concentration determination
Example 26
Accuracy test
A control solution was prepared according to the method of example 22 and measured according to the same chromatographic conditions as in example 22: injecting 10 μ L of the solution of cefradine into a liquid chromatograph; measuring 6 times by the same method, and recording chromatogram of the reference substance as system applicability experiment.
Weighing 3 parts of cefradine reference substances of 20mg, 25mg and 30mg respectively, placing the cefradine reference substances in 50mL volumetric flasks respectively, adding a mobile phase for dissolving and diluting to a scale, shaking up, and respectively corresponding to 80%, 100% and 120% of the concentration of the test sample. And respectively injecting 10 mu L of the mixture into a liquid chromatograph, respectively recording chromatograms, calculating by peak area according to an external standard method, and taking the ratio of the content to a theoretical value as a recovery rate, wherein the result is shown in table 5.
TABLE 5 test results on accuracy of cefradine concentration determination
The test result shows that: the concentration of the sample is in the range of 80-120%, the recovery rate after 9 times is in the range of 98-102%, the RSD value is 0.8%, is less than the limit of 2.0%, and the stability of the solution in example 27 is in accordance with the standard regulation
1. Stability at room temperature
Precisely measuring about 1mL of the sample liquid, placing the sample liquid in a 50mL volumetric flask, adding the mobile phase for dissolving, diluting to a scale, and shaking up. The solution was left at room temperature for 0, 2, 4, 6, 8, 10, and 12 hours, and the stability of the solution was examined by the measurement method of example 1.
Stability at 2.4 deg.C
Precisely measuring about 1mL of a material liquid sample, placing the sample in a 50mL volumetric flask, adding a mobile phase for dissolving, diluting to a scale, and shaking up. The test was performed by the measurement method of example 1 after leaving at 4 ℃ for 0, 2, 4, 6, 8, 10, and 12 hours to examine the stability of the solution.
The results of the stability tests of the test solutions at room temperature and 4 ℃ are shown in Table 6.
TABLE 6 stability study of cephradine concentration solutions
As can be seen from Table 6, the RSD values of the test solutions were less than the limit of 2.0% for 7 data at room temperature and 4 deg.C, respectively. The test result shows that the sample content of the solution is stable within 12 hours under the conditions of room temperature and 4 ℃.
Example 28
Durability examination
The durability of the analysis method was examined by changing the flow rate, column temperature, detection wavelength, volume ratio of mobile phase, and different types of chromatography columns.
1. Regulating the volume ratio of 3.86 percent sodium acetate solution to 4 percent acetic acid solution in the mobile phase
By adjusting the volume ratio of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution in the mobile phase, the ratio of water: methanol 3.86% sodium acetate solution 4% acetic acid solution =3710:1200: 70:20, 3710:1200:75: 25, 3710: the test results of the cefradine test sample with the mobile phase of 1200:80:10 are shown in the table 7-1.
TABLE 7-1 determination of the durability of the HPLC method (ratio by volume of 3.86% sodium acetate solution to 4% acetic acid solution in the mobile phase)
The test results show that the separation degree of the cefradine and the adjacent peak is more than 1.5, the system applicability meets the regulation, and the RSD value of 6 data is less than 2.0%. From this, it was found that a slight change in the volume ratio of the 3.86% sodium acetate solution to the 4% acetic acid solution in the mobile phase did not affect the measurement of the cephradine concentration and the synthetase activity.
2. The durability of the analysis method was examined by varying the flow rate, column temperature, detection wavelength and different types of chromatography columns.
2.1. Changing chromatographic columns
By replacing the chromatographic column, C18 chromatographic columns of different types of Dima, agilent and Datielaite are respectively adopted to detect the sample to be tested, and the experimental result is shown in a table 7-2.
TABLE 7-2 sample assay HPLC method durability (different model chromatographic columns)
Test results show that the separation degree of the sample and adjacent peaks is larger than 1.5, the system applicability meets the specification, and the RSD value of 6 data is smaller than 2.0%. It is clear from this that different types of columns do not affect the measurement of sample concentration and synthetase activity.
2.2. Changing the detection wavelength
The detection wavelength is adjusted to be within the range of (254 +/-5 nm), sample samples are detected by adopting the detection wavelengths of 249nm, 254nm and 259nm respectively, and the experimental result is shown in a table 7-3.
TABLE 7-3 sample assay durability of HPLC method (different detection wavelength)
Test results show that the separation degree of the sample and adjacent peaks is larger than 1.5, the system applicability meets the regulation, and the RSD value of 6 data is smaller than 2.0%. From this, it was found that the measurement of the sample concentration and the synthetase activity was not affected by the different detection wavelengths.
2.3. Changing the column temperature
The column temperature was varied between (25. + -. 5 ℃ C.) and the column temperature was used to test the sample samples at 20 ℃ C., 25 ℃ C., 30 ℃ C. Respectively, and the results are shown in tables 7-4.
TABLE 7-4 sample assay HPLC method durability (different column temperatures)
Test results show that the separation degree of the sample and adjacent peaks is larger than 1.5, the system applicability meets the regulations, and the RSD value of 6 data is smaller than 2.0%. From this, it was found that a slight change in the column temperature did not affect the measurement of the sample concentration and the synthetase activity.
2.4. Varying the flow rate
The sample test sample was tested by varying the flow rate between (1.0. + -. 0.2 mL/min) with flow rates of 0.8mL/min, 1.0mL/min, 1.2mL/min, respectively, and the results are shown in tables 7-5.
TABLE 7-5 sample assay HPLC method durability (different flow rates)
Test results show that the separation degrees of the sample and adjacent peaks are larger than each other, the applicability of the system meets the regulations, and the RSD value of 6 data is smaller than each other. From this, it was found that a slight change in the flow rate did not affect the measurement of the sample concentration and the synthetase activity.
Example 29
Specificity test
Respectively carrying out destructive tests on the sample under the conditions of high temperature, illumination, acid, alkali and oxidation, and inspecting the purity of a main peak and the separation degree of the main peak and an adjacent peak of the destroyed sample; and taking a blank solvent or the damaged blank solvent for sample injection, and inspecting whether the blank solvent interferes with the determination of the content of the sample.
1. Destructive test
And (3) no damage: precisely measuring 1mL of the feed liquid, placing the feed liquid in a 50mL volumetric flask, adding the mobile phase for dissolving to a scale, shaking uniformly, carrying out sample introduction detection, and recording a chromatogram map.
a. Alkali breakdown test
Precisely measuring 1mL of feed liquid, placing the feed liquid in a 50mL volumetric flask, adding 20mL of mobile phase, adding 5mL of 0.5mol/L sodium hydroxide solution, standing for 10 minutes, adding 5mL of 0.5mol/L hydrochloric acid solution for neutralization, adding the mobile phase for dissolving and diluting to a scale, shaking uniformly, carrying out sample injection detection, and recording a chromatogram.
b. Acid breakdown test
Precisely measuring 1mL of feed liquid, placing the feed liquid in a 50mL volumetric flask, adding 20mL of mobile phase, adding 10mL of 4mol/L hydrochloric acid solution, placing the feed liquid for 20 minutes, adding 10mL of 4mol/L sodium hydroxide solution for neutralization, adding the mobile phase for dissolving and diluting to a scale, shaking uniformly, carrying out sample injection detection, and recording a chromatogram.
c. High temperature failure test
Precisely measuring 1mL of the feed liquid, placing in a 50mL volumetric flask, adding the mobile phase to dissolve and dilute to about 3/4 of the volume of the volumetric flask, heating in a water bath at 80 ℃ for 15 minutes, cooling and cooling to room temperature. Adding mobile phase, dissolving and diluting to scale, shaking, detecting by sample injection, and recording chromatogram.
d. Oxidative destruction test
Precisely measuring 1mL of feed liquid, placing in a 50mL volumetric flask, adding 20mL of mobile phase, adding 5mL of 30% hydrogen peroxide, placing for 20min, adding mobile phase for dissolving and diluting to scale, shaking uniformly, detecting by sample injection, and recording chromatogram.
e. Light damage test
Precisely measuring 1mL of the feed liquid, placing the feed liquid in a 50mL volumetric flask, adding a mobile phase for dissolving to a scale, placing the solution in a 4500lx illumination test box for 24 hours, taking out the solution, shaking uniformly, carrying out sample injection detection, and recording a chromatogram.
The results of the content measurement of the above samples subjected to the non-destructive, alkali destructive, acid destructive, high temperature destructive, oxidation destructive, and light destructive tests are shown in Table 8.
TABLE 8 sample assay specificity failure results
Note: the main peak area is converted from the peak area measured by an undamaged peak area sample according to the sample weighing and is a theoretical value; the destruction degree is the percentage of the area of the main peak after destruction to the area of the main peak before destruction; and R is the separation degree of the main peak area and the impurity peak of the front adjacent peak.
Test results show that all the damage degrees reach 80-90%, and the main peak area and the impurity peak separation degree of the front adjacent peak are all larger than the specified value. From this, it was found that the main peak was a single peak, and the content of the sample was not affected by the degradation impurities generated under each destruction condition.
Claims (6)
1. A method for analyzing activity of cefradine synthetase is characterized in that after the content of cefradine in a reaction solution to be detected is measured by adopting a high performance liquid chromatography, the activity of the synthetase is obtained by calculation, and the method comprises the following specific steps:
(1) Preparation of control solutions: precisely weighing a cefradine reference substance, adding a mobile phase for dissolving, quantitatively diluting to prepare a cefradine reference substance solution with the concentration of 0.05-1.0 mg/mL, and shaking up;
(2) Preparation of a sample solution to be tested:
preparation of a substrate solution: weighing 4.3g of 7-ADCA and 4.1g of dihydroglycine methyl ester hydrochloride, adding 200ml of purified water for dissolving, and adjusting the pH value to be =7.0 by using ammonia water for later use;
preparing cefradine synthetase: taking a proper amount of cefradine enzyme, washing with 100ml of purified water, carrying out suction filtration until no water drops, and precisely weighing 2.0g for later use;
preparing a reaction solution sample: starting a water area constant-temperature oscillating screen, setting the temperature to be 20 ℃, oscillating the water area constant-temperature oscillating screen at the oscillation frequency of 265, accurately measuring 90ml of substrate solution, placing the substrate solution in a 250 conical flask, oscillating the substrate solution for 10min to ensure that the temperature of the solution is 20 ℃, adding 2.0g of cefradine synthetase, washing the bottleneck with 10ml of purified water, timing for 10min, and keeping the timing for later use;
preparing a solution of a sample to be detected: precisely measuring a reaction solution sample, adding a mobile phase for dissolving, quantitatively diluting to prepare a to-be-measured sample solution with the concentration of 0.05-1.0 mg/mL, and shaking up;
(3) Analysis of synthetase Activity: respectively and precisely measuring 10 mu L of each of the solution of the sample to be measured and the solution of the reference substance, injecting the solution of the sample to be measured and the solution of the reference substance into a high performance liquid chromatograph, recording chromatograms of the sample to be measured and the reference substance, and calculating by peak area according to an external standard method to obtain the concentration of cefradine in the sample to be measured, thereby calculating the activity of the synthetase, wherein the calculation formula is as follows:
enzyme activity calculation formula:
wherein f is a correction factor; a is the concentration (mmol/L) of the cefradine in the reaction solution, t is the reaction time (min), m is the mass (g) of the enzyme sample, the molecular weight of the cefradine is 349.41 (without water), and U is the activity (mmol/min) of the enzyme.
2. The method for assaying the activity of cefradine synthase according to claim 1, wherein the chromatographic conditions are: octadecylsilane chemically bonded silica is used as a filling agent, the flow rate of a mobile phase is 0.8-1.2 mL/min, the detection wavelength is 249-259 nmnm, the column temperature is 20-30 ℃, and the number of theoretical plates is not less than 2000 calculated according to a main peak.
3. The method for analyzing the activity of cefradine synthase according to claim 1 or 2, wherein the mobile phase is a mixed solution of water, methanol, 3.86% sodium acetate solution and 4% acetic acid solution, and the volume ratio of the water, the methanol, the 3.86% sodium acetate solution and the 4% acetic acid solution is 3710:1200: 70-80: 14-16.
4. The method for analyzing the activity of cefradine synthase according to claim 2, wherein the 3.86% sodium acetate solution is prepared by: weighing 64.0g of sodium acetate trihydrate, dissolving in 500ml of ultrapure water, and adding ultrapure water to dilute to 1000ml after dissolving;
the preparation method of the 4% acetic acid solution comprises the following steps: measuring 9.6ml of glacial acetic acid, dissolving in 250ml of ultrapure water, and uniformly mixing.
5. The method for assaying the activity of cefradine synthase according to claim 2, wherein the flow rate of the mobile phase is 1.0mL/min;
the column temperature is 25 ℃;
the detection wavelength is 254nm.
6. The method for assaying the activity of cefradine synthase according to claim 1, wherein the concentration of the control solution and the concentration of the test sample solution in step (3) are 0.5mg/mL.
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| CN1813069A (en) * | 2003-07-03 | 2006-08-02 | 帝斯曼知识产权资产管理有限公司 | Process for the preparation of cephradine |
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