Method for detecting penicillin acylase in cefaclor raw material
Patent Information
- Application Number
- CN202511089899.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-05
- Publication Date
- 2025-11-07
Smart Images
Figure CN120908336A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of medical detection, and particularly relates to a method for detecting penicillin acylase in cefaclor raw materials. BACKGROUND
[0002] Penicillin acylase is an important enzyme in the antibiotic industry, which can hydrolyze penicillin in reverse to produce 6-aminopenicillanic acid (6-APA) and 7-amino-deacetoxycephalosporanic acid (7-ADCA); at the same time, penicillin acylase can catalyze the condensation of levoglutamine methyl ester and 7-amino-3-chlorocephalosporanic acid to generate cefaclor. Since the 1970s, penicillin acylase research and production have been carried out in China, and most of the penicillin acylase determination methods use NIPAB method (2-nitro-5-phenylacetamidobenzoic acid) and alkali titration method. Among them, the alkali titration method usually uses the hydroxylamine method and the iodometric method.
[0003] However, when the alkali titration method is used to determine penicillin acylase, the penicillin contained therein may interfere with the reaction, and the substrate penicillin needs to be removed during determination. In the operation process of the alkali titration method, while timing, the pH value needs to be maintained at about 8.0, and the experiment operation needs the attention of two people to assist each other, which is complicated and has low sensitivity; meanwhile, the method also has certain errors in dilution and determination of titration end point. The NIPAB method overcomes the above-mentioned shortcomings to some extent, but cannot reflect the action relationship between the enzyme and its real substrate penicillin, and has poor stability and low data accuracy. SUMMARY
[0004] The purpose of the present application is to provide a method for detecting penicillin acylase in cefaclor raw materials to overcome the shortcomings of the prior art, and the technical scheme is as follows:
[0005] A method for detecting penicillin acylase in cefaclor raw materials comprises the following steps:
[0006] (1) Preparation of the test sample solution: an appropriate amount of obtained cefaclor raw material is dissolved in a solvent and quantitatively diluted to prepare a test sample solution;
[0007] (2) Preparation of the control sample solution: an appropriate amount of penicillin G acylase is dissolved in a solvent and quantitatively diluted to prepare a control sample solution;
[0008] (3) Precise measurement of the above-mentioned solution is directly detected by using a liquid chromatograph-fluorescence detector, and a chromatogram is recorded, and the content of penicillin acylase is calculated according to the chromatogram by using an external standard method,
[0009] The high performance liquid chromatography detection condition is that: the spherical hydrophilic silica gel is used as the filler of the chromatographic column; water-acetonitrile-trifluoroacetic acid (950:50:5) is used as the mobile phase A, and water-acetonitrile-trifluoroacetic acid (100:900:5) is used as the mobile phase B, gradient elution is carried out, and the gradient elution condition is that:
[0010] The first gradient elution time is 0-29 minutes, the proportion of the mobile phase A is 52%-35%, and the proportion of the mobile phase B is 48%-65%;
[0011] The second gradient elution time is 29-30 minutes, the proportion of the mobile phase A is 35%-52%, and the proportion of the mobile phase B is 65%-48%;
[0012] The third gradient elution time is 30-35 minutes, the proportion of the mobile phase A is 52%, and the proportion of the mobile phase B is 48%.
[0013] Preferably, the preparation steps of the test solution are as follows: 0.5 g of cefaclor raw material is accurately weighed, 5 ml of phosphate buffer is added for dilution, 1.0 mol / L sodium hydroxide solution is added dropwise for oscillation until complete dissolution, the phosphate buffer is added for dilution to the mark, and the mixture is shaken and centrifuged (6000 r / min, 5 min) to obtain the supernatant.
[0014] Further preferably, the preparation steps of the phosphate buffer are as follows: about 6.8 g of potassium dihydrogen phosphate is weighed, 29 ml of 1.0 mol / L sodium hydroxide solution is added, water is used for dissolution and dilution to 1000 ml, and the pH value is adjusted to 7.0.
[0015] Preferably, the preparation steps of the control solution are as follows: an appropriate amount of penicillin G acylase control is accurately weighed, the phosphate buffer is added for quantitative dilution to prepare a solution containing about 0.5 μg of penicillin G acylase per 1 ml.
[0016] Preferably, the chromatographic column is a TSK gel Octadecyl-4PW, the length of the chromatographic column is 15 cm, the diameter of the chromatographic column is 4.6 mm, and the particle size of the filler in the chromatographic column is 7 μm.
[0017] Preferably, the fluorescence detector detects at an excitation wavelength of 275 nm and an emission wavelength of 345 nm.
[0018] Preferably, in the detection method, the column temperature of the chromatographic column is 40 ℃, the injection volume is 100 μl, and the flow rate is 1.0 ml per minute.
[0019] The present application has the following beneficial effects:
[0020] The detection method of the present application uses penicillin G acylase (PGA) as the enzyme protein standard, uses a chromatographic column TSK gel Octadecyl-4PW with good separation effect on proteins for separation, and uses liquid chromatography-fluorescence detector for direct detection, thereby avoiding the defects of the existing detection methods. The method has good specificity, linear relationship, repeatability, intermediate precision, accuracy and stability; the operation is simple, the detection result is stable, and the monitoring data is accurate. PGA shows good first-order linear relationship in the range of 0.2 g / mL to 4.0 g / mL, the linear regression equation is Y=2.1962x+0.5016, and the correlation coefficient (R) is greater than 0.998, and the external standard method is used for quantification. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 High performance liquid chromatogram of the test sample solution.
[0022] Figure 2 High performance liquid chromatogram of the blank solution.
[0023] Figure 3 High performance liquid chromatogram of the control solution. DETAILED DESCRIPTION
[0024] The technical solutions of the present application will be described in detail below in combination with the embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.
[0025] It should be noted that the experimental methods used in the implementation examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.
[0026] In the present application, unless otherwise specified, the numerical range "a~b" represents a shorthand representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0~5" represents that all real numbers between "0~5" have been listed herein, and "0~5" is only a shorthand representation of these numerical combinations.
[0027] In the present application, all the embodiments and preferred embodiments mentioned in the present application can be combined to form new technical solutions if not otherwise specified.
[0028] In the present application, if not otherwise specified, all the steps mentioned herein can be carried out in sequence or randomly, but preferably in sequence; for example, the method comprises steps (a) and (b), which means that the method can comprise steps (a) and (b) in sequence, or steps (b) and (a) in sequence; for example, the method mentioned herein can further comprise step (c), which means that step (c) can be added to the method in any sequence; for example, the method can comprise steps (a), (b) and (c), or steps (a), (c) and (b), or steps (c), (a) and (b), etc.
[0029] In the present application, if not otherwise specified, the specific values and specific substances in the embodiments of the present application can be combined with other features described in the description; for example, if the description mentions that the reaction temperature is 10-100°C, and the embodiment mentions that the reaction temperature is 20°C, then it can be considered that the present application has specifically disclosed the range of 10-20°C, or the range of 20-100°C, and the range can be combined with other features described in the description to form a new technical solution.
[0030] Example 1
[0031] Preparation of test solution: 0.5 g of the product was accurately weighed into a 10 ml volumetric flask, diluted with 5 ml of phosphate buffer (about 6.8 g of potassium dihydrogen phosphate was weighed, 29 ml of 1.0 mol / L sodium hydroxide solution was added, dissolved and diluted with water to 1000 ml, and the pH value was adjusted to 7.0), 1.0 mol / L sodium hydroxide solution was added dropwise, and the solution was shaken until it was completely dissolved. The phosphate buffer was added to the mark, shaken well, and centrifuged (6000 rpm for 5 minutes). The supernatant was taken.
[0032] Preparation of reference solution: an appropriate amount of penicillin G acylase reference substance was accurately weighed, diluted with phosphate buffer to prepare a solution containing about 0.5 μg of penicillin G acylase per 1 ml.
[0033] Chromatographic conditions: spherical hydrophilic silica gel as filler (TSKgel Octadecyl-4PW, 15 cm x 4.6 mm, 7 μm or a chromatographic column with equivalent performance); water-acetonitrile-trifluoroacetic acid (950:50:5) as mobile phase A, water-acetonitrile-trifluoroacetic acid (100:900:5) as mobile phase B, gradient elution according to Table 1; fluorescence detector for detection, excitation wavelength 275 nm, emission wavelength 345 nm; flow rate 1.0 ml / min; column temperature 40°C; injection volume 100 μl.
[0034] Table 1 Gradient elution conditions
[0035]
[0036] Example 2
[0037] Linearity experiment
[0038] The control solution was taken as the linear test solution, and was detected by using the high performance liquid chromatograph under the chromatographic conditions given in the example. The chromatogram was recorded, and the linear regression of the concentration and the peak area was performed. The experimental results are shown in Table 2.
[0039] Table 2 Linearity experiment results
[0040]
[0041]
[0042] Example 3
[0043] Repeatability experiment
[0044] The test solution was injected 6 times, and was tested by using the high performance liquid chromatograph. The repeatability was calculated by using the peak area. The experimental results are shown in Table 3.
[0045] Table 3
[0046]
[0047] Example 4
[0048] Precision experiment
[0049] The control solution was taken, and was injected 5 times. The chromatogram was recorded (Table 4). The experimental results are shown in Table 4. Figure 3
[0050] Table 4
[0051]
[0052] The results show that the RSD (n=5) of the peak area of cefaclor is 0.559%, and the RSD (n=5) of the retention time of the main peak is 1.230%, which indicates that the precision of the method is good.
[0053] Example 5
[0054] Stability experiment
[0055] The test solution was taken, and was injected after being placed at room temperature for 0, 1, 2, 4, 6, 8, 12, 16 and 24 hours. The results show that the RSD of the peak area of cefaclor is 0.36%, which indicates that the test solution is stable at room temperature within 24 hours.
[0056] The application provides a method for detecting the content of penicillin G acylase in cefaclor. The method has good specificity, linear relationship, repeatability, intermediate precision, accuracy and stability; and the method is simple and has strong operability. PGA has good first-order linear relationship in the range of 0.2g / mL-4.0g / mL, the linear regression equation is Y=2.1962x+0.5016, the correlation coefficient (R) is greater than 0.998, and the method meets the requirements for external standard quantification.
[0057] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the application, but not to limit them; although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the application, and they should be covered in the scope of the claims and the description of the application.
Claims
1. A method for detecting penicillin acylase in cefaclor raw material, characterized by, It comprises the following steps: (1) Preparation of the test solution: take the obtained cefaclor raw material, dissolve it in a solvent and dilute it quantitatively to prepare a test solution; (2) Preparation of the control solution: take penicillin G acylase, dissolve it in a solvent and dilute it quantitatively to prepare a control solution; (3) Directly detect the above-mentioned solutions by using liquid chromatography-fluorescence detector, record the chromatogram, and calculate the content of penicillin acylase according to the chromatogram by using the external standard method, The conditions for high-performance liquid chromatography detection are as follows: the chromatographic column is filled with spherical hydrophilic silica gel; water-acetonitrile-trifluoroacetic acid (950:50:5) is used as mobile phase A, and water-acetonitrile-trifluoroacetic acid (100:900:5) is used as mobile phase B for gradient elution, and the gradient elution conditions are as follows: The first gradient elution time is 0-29 minutes, the proportion of mobile phase A is 52%-35%, and the proportion of mobile phase B is 48%-65%; The second gradient elution time is 29-30 minutes, the proportion of mobile phase A is 35%-52%, and the proportion of mobile phase B is 65%-48%; The third gradient elution time is 30-35 minutes, the proportion of mobile phase A is 52%, and the proportion of mobile phase B is 48%.
2. The method for detecting penicillin acylase in cefaclor raw material according to claim 1, characterized in that, The specific steps for preparing the test solution are as follows: take 0.5g of cefaclor raw material, accurately weigh it, place it in a 10ml volumetric flask, dilute it with 5ml of phosphate buffer, drop 1.0mol / L sodium hydroxide solution while oscillating until completely dissolved, dilute it with phosphate buffer to the mark, shake well, centrifuge (6000rpm / min, 5 minutes), and take the supernatant.
3. The method for detecting penicillin acylase in cefaclor raw material according to claim 2, characterized in that, The preparation steps of the phosphate buffer are as follows: take about 6.8g of potassium dihydrogen phosphate, add 29ml of 1.0mol / L sodium hydroxide solution, dissolve and dilute it with water to 1000ml, and adjust the pH value to 7.
0.
4. The method for detecting penicillin acylase in cefaclor raw material according to claim 1, characterized in that, The specific steps for preparing the control solution are as follows: take an appropriate amount of penicillin G acylase control product, accurately weigh it, dilute it with phosphate buffer to prepare a solution containing about 0.5ug of penicillin G acylase per 1ml.
5. The method for detecting penicillin acylase in cefaclor raw material according to claim 1, characterized in that, The chromatographic column is TSK gel Octadecyl-4PW, the length of the chromatographic column is 15cm, the diameter of the chromatographic column is 4.6mm, and the particle size of the filler in the chromatographic column is 7um.
6. The method for detecting penicillin acylase in cefaclor raw material according to claim 1, characterized in that, The fluorescence detector detects at an excitation wavelength of 275nm and an emission wavelength of 345nm.
7. The method for detecting penicillin acylase in cefaclor raw material according to claim 1, characterized in that, In the detection method, the column temperature of the chromatographic column is 40℃, the injection volume is 100ul, and the flow rate is 1.0ml per minute.