CN113567607A

CN113567607A - Method for detecting content of penicillin G acylase in cefaclor

Info

Publication number: CN113567607A
Application number: CN202110837459.7A
Authority: CN
Inventors: 王炳玲; 冯玉蓉; 张�浩
Original assignee: Hebei Ji'an Testing Service Co ltd
Current assignee: Hebei Ji'an Testing Service Co ltd
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2021-10-29

Abstract

The invention belongs to the technical field of substance detection. The invention provides a method for detecting the content of penicillin G acylase in cefaclor, which comprises the preparation of a reference substance intermediate solution, reference substance solutions with different gradient concentrations, a test substance solution and a test substance labeling solution; detecting the reference solution by adopting a liquid chromatograph, and obtaining a linear equation according to an external standard method; and detecting the test sample solution and the test sample standard solution by adopting a liquid chromatograph, and obtaining the content of the penicillin G acylase in the test sample solution and the test sample standard solution according to a linear equation and the measured peak area. The detection limit of the method is 2.0mg/kg, and the quantification limit is 4.0 mg/kg; the method for detecting the content of the enzyme protein has good specificity, linear relation, repeatability, intermediate precision, accuracy, stability and durability; and the method is simple and convenient and has strong operability.

Description

Method for detecting content of penicillin G acylase in cefaclor

Technical Field

The invention relates to the technical field of substance detection, in particular to a method for detecting the content of penicillin G acylase in cefaclor.

Background

At present, the method for detecting the residual amount of penicillin G acylase enzyme protein in cefaclor raw materials mainly comprises a liquid chromatography-tandem mass spectrometry method. The detection limit of the method is 2.0mg/kg, the quantification limit is 4.0mg/kg, and the method can be used for absolute quantitative detection of protein content with high sensitivity and high selectivity. The method is characterized in that the method comprises the steps of carrying out enzymolysis on Penicillin G Acylase (PGA) to detect a characteristic peptide segment, and synthesizing an isotope labeled internal standard of the characteristic peptide segment to overcome the matrix effect of LC-MS/MS so as to carry out quantitative analysis.

However, the liquid chromatography tandem mass spectrometry requires identification and analysis of characteristic peptide fragments, and is complex to operate. Therefore, the research and development of the method for detecting the residual amount of the penicillin G acylase enzyme protein in the cefaclor raw material, which is simple in operation, good in detection effect, real and reliable, has important value and significance.

Disclosure of Invention

The invention aims to provide a method for detecting the content of penicillin G acylase in cefaclor aiming at the defects of the prior art. The method for detecting the content of the enzyme protein has good specificity, linear relation, repeatability, intermediate precision, accuracy, stability and durability; and the method is simple and convenient and has strong operability.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for detecting the content of penicillin G acylase in cefaclor, which comprises the following steps:

1) mixing a penicillin G acylase reference substance with a phosphate buffer solution to obtain a reference substance intermediate solution;

2) diluting the intermediate solution of the reference substance by using a phosphate buffer solution to obtain reference substance solutions with different gradient concentrations;

3) mixing a test sample cefaclor, a phosphate buffer solution and a sodium hydroxide solution, and centrifuging to obtain a test sample solution;

4) mixing a test sample cefaclor, a reference intermediate solution, a phosphate buffer solution and a sodium hydroxide solution, and centrifuging to obtain a test sample labeling solution;

5) detecting the reference solution by adopting a liquid chromatograph, and obtaining a linear equation according to an external standard method;

6) and detecting the test sample solution and the test sample standard solution by adopting a liquid chromatograph, and obtaining the content of the penicillin G acylase in the test sample solution and the test sample standard solution according to a linear equation and the measured peak area.

Preferably, the phosphate buffer solution in the steps 1) to 4) is prepared from monopotassium phosphate, a sodium hydroxide solution and water, the mass volume ratio of the monopotassium phosphate to the sodium hydroxide solution to the water is 6-7.5 g: 25-35 mL: 950-1000 mL, and the concentration of the sodium hydroxide solution is 0.8-1.2 mol/L.

Preferably, the concentration of penicillin G acylase in the intermediate solution of the reference substance in the step 1) is 45-55 mu G/mL; in the penicillin G acylase reference substance, the total protein content is 11-13 mg/mL.

Preferably, the gradient concentrations of penicillin G acylase in said control solution of step 2) are 0.1. mu.g/mL, 0.2. mu.g/mL, 0.5. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL and 5.0. mu.g/mL, respectively.

Preferably, the mass-to-volume ratio of the sample cefaclor, the phosphate buffer solution and the sodium hydroxide solution in the step 3) is 0.4-0.6 g: 8-10 mL: 0.5-3 mL;

and 4) the mass-to-volume ratio of the test cefaclor to be tested, the intermediate solution of the reference substance, the phosphate buffer solution and the sodium hydroxide solution is 0.4-0.6 g: 0.05-0.2 mL: 7-9.5 mL: 0.5-3 mL.

Preferably, in the steps 3) and 4), the concentration of the sodium hydroxide solution is 0.8-1.2 mol/L, the rotation speed of the centrifugal treatment is 5500-6500 r/min, and the time is 3-8 min.

Preferably, in the detection process of the step 5) and the step 6), gradient elution is carried out by adopting a mixed solution of a mobile phase A and a mobile phase B; in the mixed solution, the volume ratio of the mobile phase A to the mobile phase B is 35-60: 40-65; the time of gradient elution is less than or equal to 1 h.

Preferably, the mobile phase a and the mobile phase B each comprise water, acetonitrile and trifluoroacetic acid; in the mobile phase A, the volume ratio of water to acetonitrile to trifluoroacetic acid is 940-960: 45-55: 4-6, and in the mobile phase B, the volume ratio of water to acetonitrile to trifluoroacetic acid is 95-105: 880-920: 4-6.

Preferably, in the detection process of the step 5) and the step 6), the flow rate is 0.8-1.2 mL/min, the column temperature is 35-45 ℃, and the sample injection amount is 50-100 mu L; the excitation wavelength is 270-280 nm, and the emission wavelength is 340-350 nm.

The invention has the beneficial effects that:

the method for determining the residual quantity of the penicillin G acylase in the cefaclor raw material by adopting the liquid chromatography is characterized in that the penicillin G acylase is used as an enzyme protein standard substance, the detection is carried out by using a liquid chromatography-fluorescence detector, the PGA has a good first-order linear relation in the range of 0.2-5.0 mu G/mL, the detection limit of the method is 2.0mg/kg, and the quantification limit is 4.0 mg/kg. The method for detecting the content of the enzyme protein has good specificity, linear relation, repeatability, intermediate precision, accuracy, stability and durability; and the method is simple and convenient and has strong operability.

Drawings

FIG. 1 is a graph of a bovine serum albumin standard solution of example 1;

FIG. 2 is an enzyme protein chromatogram detected by the UV detector and the fluorescence detector of example 1, wherein the chromatogram corresponds to a 200nm spectrum of the UV detector, a 280nm spectrum of the UV detector, and a fluorescence detector from top to bottom;

FIG. 3 is a typical chromatogram of a control solution of example 2;

fig. 4 is a chromatogram showing the specificity of the blank solution, the reference solution, the sample solution and the sample solution in example 3, wherein the blank solution, the reference solution, the sample solution and the sample solution correspond to each other from top to bottom.

Detailed Description

The phosphate buffer solution in the steps 1) to 4) is preferably prepared from monopotassium phosphate, a sodium hydroxide solution and water, and the mass-to-volume ratio of the monopotassium phosphate, the sodium hydroxide solution and the water is preferably 6-7.5 g: 25-35 mL: 950-1000 mL, more preferably 6.5-7 g: 28-32 mL: 960-980 mL, and even more preferably 7g:30mL:970 mL; the concentration of the sodium hydroxide solution is preferably 0.8-1.2 mol/L, and more preferably 1 mol/L.

The concentration of penicillin G acylase in the intermediate solution of the reference substance in the step 1) is preferably 45-55 mug/mL, and more preferably 50 mug/mL; in the penicillin G acylase control, the total protein content is preferably 11-13 mg/mL, and more preferably 12 mg/mL.

The gradient concentrations of penicillin G acylase in the control solution of step 2) of the present invention are preferably 0.1. mu.g/mL, 0.2. mu.g/mL, 0.5. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL and 5.0. mu.g/mL, respectively.

The mass-volume ratio of the test sample cefaclor, the phosphate buffer solution and the sodium hydroxide solution in the step 3) is preferably 0.4-0.6 g: 8-10 mL: 0.5 to 3mL, more preferably 0.5 g: 9mL of: 1-2.5 mL, more preferably 0.5 g: 9mL of: 2 mL.

The mass-to-volume ratio of the test cefaclor, the reference intermediate solution, the phosphate buffer solution and the sodium hydroxide solution in the step 4) is preferably 0.4-0.6 g: 0.05-0.2 mL: 7-9.5 mL: 0.5 to 3mL, more preferably 0.5 g: 0.1-0.15 mL: 7.5-9 mL: 1-2.5 mL, more preferably 0.5 g: 0.1 mL: 8mL of: 2 mL.

In steps 3) and 4) of the invention, the concentration of the sodium hydroxide solution is preferably 0.8-1.2 mol/L, and more preferably 1 mol/L; the rotation speed of the centrifugal treatment is preferably 5500-6500 r/min, more preferably 5700-6200 r/min, and more preferably 5900-6000 r/min; the time for the centrifugal treatment is preferably 3-8 min, more preferably 4-7 min, and even more preferably 5-6 min.

In the detection process of the step 5) and the step 6), mixed solution of a mobile phase A and a mobile phase B is adopted for gradient elution; in the mixed solution, the volume ratio of the mobile phase A to the mobile phase B is preferably 35-60: 40-65, more preferably 40-50: 45-60, and even more preferably 45: 50-55; the time of gradient elution is preferably less than or equal to 1 hour, and more preferably 10-40 min.

The mobile phase A and the mobile phase B of the invention preferably both comprise water, acetonitrile and trifluoroacetic acid; in the mobile phase A, the volume ratio of water to acetonitrile to trifluoroacetic acid is preferably 940-960: 45-55: 4-6, more preferably 950: 48-52: 5, and even more preferably 950:50: 5; in the mobile phase B, the volume ratio of water to acetonitrile to trifluoroacetic acid is preferably 95-105: 880-920: 4-6, more preferably 100: 890-910: 5, and even more preferably 100:900: 5.

In the detection process of the steps 5) and 6), the flow rate is preferably 0.8-1.2 mL/min, more preferably 0.9-1.1 mL/min, and even more preferably 1 mL/min; the column temperature is preferably 35-45 ℃, more preferably 37-42 ℃, and more preferably 38-40 ℃; the sample injection amount is preferably 50-100 muL, more preferably 60-90 muL, and even more preferably 70-80 muL; the excitation wavelength is preferably 270-280 nm, more preferably 273-278 nm, and even more preferably 275 nm; the emission wavelength is preferably 340-350 nm, more preferably 343-347 nm, and even more preferably 345 nm.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The instrument models, specifications and manufacturers used in the examples are as follows:

name of chromatographic column: TSKgel octadecenyl-4 PW, Specification: 15cm × 4.6mm,7 μm, manufacturer: eastern Cao; mobile phase A: a mixed solution of water, acetonitrile and trifluoroacetic acid in a volume ratio of 950:50:5, a mobile phase B: a mixed solution of water, acetonitrile and trifluoroacetic acid in a volume ratio of 100:900: 5; flow rate: 1 mL/min; column temperature: at 40 ℃.

Liquid chromatograph: the model is as follows: thermo UltiMate3000, manufacturer: saimer Feishale science.

One tenth of a ten thousand electronic balance: the model is as follows: MS 105DU, manufacturer: the mertler.

A fluorescence detector: the excitation wavelength is 275nm, and the emission wavelength is 345 nm; an ultraviolet detector: the detection wavelength is 200nm and 280 nm.

Desk-top high-speed refrigerated centrifuge: the model is as follows: TGL16M, manufacturer: kate instruments Ltd.

Ultraviolet-visible spectrophotometer: the model is as follows: TU 1810, manufacturer: beijing Puproeutro general instruments, Inc.;

small vortex mixer: the model is as follows: MX-S, manufacturer: SCILOGEX.

Bradford protein concentration determination kit (product content: 5 XG 250 staining solution; PBS dilution): the manufacturer: solarbio, lot number: 20191031.

the specifications, manufacturers and lot numbers of the reagents used in the examples are as follows:

trifluoroacetic acid: specification: HPLC, manufacturer: mclin, lot number: C10919649.

acetonitrile: specification: HPLC, manufacturer: sunrise chem, INC, batch number: 19031502.

potassium dihydrogen phosphate: specification: AR, manufacturer: tianjin Guangfu Fine chemical research institute, lot number: 2018.7.28.

sodium hydroxide: specification: AR, manufacturer: tianjin Guangfu Fine chemical research institute, lot number: 2017.5.26.

water: the manufacturer: the laboratory pure water system (Zhi ang) is a laboratory first-grade water system.

Bovine Serum Albumin (BSA): the manufacturer: solarbio, lot number: 20191029, concentration: 5 mg/mL.

Preparation of Phosphate Buffered Saline (PBS): 6.80g of potassium dihydrogen phosphate was weighed, 29mL of 1.0mol/L sodium hydroxide solution was added, and the mixture was diluted with water to 1000mL, and the pH was adjusted to 7.0.

And (3) testing the sample: cefaclor raw material, batch number: 22051906002.

preparation of bovine serum albumin standard solution: BSA control was diluted with phosphate buffer to make a solution with a concentration of 0.2 mg/mL.

Preparation of penicillin G acylase to be tested: precisely measuring 0.1mL of penicillin G acylase solution to be calibrated, diluting the solution to 1mL by using PBS diluent, and shaking the solution uniformly for later use.

Preparation of coomassie brilliant blue reagent: mixing 5 XG 250 staining solution with a volume ratio of 1:4 with water, and shaking up for later use.

Example 1

Calibration of penicillin G acylase reference substance and determination of main component content

Measuring BSA, penicillin G acylase, PBS and Coomassie brilliant blue reagents according to the amount shown in Table 1, respectively adding the reagents into test tubes with the numbers of 1-8, uniformly mixing by vortex, standing for 3min, measuring absorbance at 595nm by adopting a Bradford kit, and calculating the total protein content of the penicillin G acylase according to a standard curve method.

TABLE 1 determination of the protein concentration of penicillin G acylase in different solutions

The bovine serum albumin content and the corrected absorbance in table 1 were used to obtain the bovine serum albumin standard solution curve, as shown in fig. 1.

From FIG. 1 the calculation formula for the total protein content of penicillin G acylase can be derived:

wherein X is the total protein content of penicillin G acylase in units: mg/mL; c is the total protein amount in 0.05mL of penicillin G acylase to be tested, unit: mu G, C is obtained by substituting 0.05mL of corrected absorbance of penicillin G acylase to be detected into a linear equation; v is the volume of the penicillin G acylase solution to be detected added into the determination solution, and is 0.05 mL; f is the dilution factor of penicillin G acylase solution, 10.

From the above calculation formula, the total protein content of penicillin G acylase was 12.0 mg/mL.

Taking the batch of penicillin G acylase as an enzyme protein reference substance, precisely measuring 0.1mL of the penicillin G acylase reference substance, diluting to 10mL by using a phosphate buffer solution, and uniformly mixing. Calculating the main component content of the penicillin G acylase of the batch by a liquid chromatography according to an area normalization method. The liquid phase gradient conditions are shown in table 2.

TABLE 2 liquid phase gradient conditions for determination of the content of major components of penicillin G acylase

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0～40	60→36	40→64
40～40.5	36→60	64→40
			40.5～45	60	40

And acquiring the spectrum of the ultraviolet detector and the spectrum of the fluorescence detector to obtain a graph 2, wherein the graph 2 is respectively a spectrum of the ultraviolet detector of 200nm, a spectrum of the ultraviolet detector of 280nm and a spectrum of the fluorescence detector from top to bottom.

As can be seen from fig. 2, the response values of the control solution in three acquisition channels are: the ultraviolet 200nm channel has no obvious response, the ultraviolet 280nm channel and the fluorescence channel have obvious responses, the reference solution has the highest response value in a fluorescence detector, three obvious peaks appear, the three peaks are marked as a peak 1, a peak 2 and a peak 3, the peak 2 is selected for quantitative analysis due to the high response value and stable signals of the peak 2, the purity of the main peak of penicillin G acylase measured twice is 73.22 percent and 73.82 percent respectively according to the calculation of a peak area normalization method, and the content of the main component of the penicillin G acylase of the batch is 8.82 mg/mL. Calculating the formula: 12.0mg/mL × (73.22% + 73.82%)/2 ═ 8.82 mg/mL.

Example 2

The residual amount of penicillin G acylase in cefaclor raw material was determined according to high performance liquid chromatography (China pharmacopoeia 2015 edition four parts general rules 0512).

Gradient elution is carried out by adopting a chromatographic column according to the gradient conditions in the table 3, 100 mu L of reference solution is precisely measured and injected into a liquid chromatograph, the recorded chromatogram should be consistent with the typical chromatogram of the reference solution in the figure 3, and the separation degree between two peaks should be more than 1.5.

TABLE 3 liquid phase gradient conditions

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0～29	52→35	48→65
29～30	35→52	65→48
			30～35	52	48

Precisely weighing 0.5g of a sample, placing the sample in a 10mL volumetric flask, adding 5mL of phosphate buffer solution for dilution, dropwise adding 1.0mol/L of sodium hydroxide solution, oscillating while dropwise adding until the sample is completely dissolved, diluting the sample with the phosphate buffer solution, fixing the volume to 10mL, uniformly mixing, centrifuging at 6000r/min for 5min, and taking supernatant as a sample solution. An appropriate amount of penicillin G acylase control was precisely measured and diluted with phosphate buffer to 0.5. mu.g/mL (peak 2) of penicillin G acylase as a control solution. Precisely measuring 100 μ L of each of the two solutions, injecting into a liquid chromatograph, and recording chromatogram. The penicillin G acylase content of cefaclor was calculated by external standard method by peak 2 area with reference to the typical chromatogram of the control solution of fig. 3.

Example 3

And (3) carrying out methodology verification on the penicillin G acylase content by adopting a liquid chromatograph, wherein the methodology verification comprises specificity, linear relation, quantitative limit and detection limit, repeatability, intermediate precision, accuracy, solution stability and durability.

And (3) specificity test:

specificity acceptance criteria: the blank solution and the sample solution have no interference to the components to be detected, and the target peak can be completely separated from other peaks.

Preparing a reference substance intermediate solution: accurately weighing 0.057mL of PGA reference substance into a 10mL volumetric flask, diluting to scale with phosphate buffer solution, shaking up, and preparing the reference substance intermediate solution with the concentration of 50 mug/mL. The control intermediate used in all the examples was the same as that used in example 3.

Preparation of a reference solution: precisely measuring 0.15mL of control intermediate solution, diluting with phosphate buffer solution, diluting to 10mL, shaking to obtain control solution with concentration of 0.75 μ g/mL.

Preparing a test solution: precisely weighing 0.5g of a test sample, diluting with 6mL of phosphate buffer solution, uniformly mixing, dropwise adding 1.0mol/L sodium hydroxide solution, oscillating while dropwise adding until the test sample is completely dissolved, fixing the volume to 10mL by using the phosphate buffer solution, uniformly mixing, centrifuging at 6000r/min for 5min, and taking supernatant as the test sample solution.

Preparing a sample labeling solution: precisely weighing 0.5g of a test sample, adding 0.15mL of intermediate solution of a reference sample, uniformly mixing, diluting with 6mL of phosphate buffer solution, uniformly mixing, dropwise adding 1.0mol/L sodium hydroxide solution while shaking until the test sample is completely dissolved, fixing the volume to 10mL by using the phosphate buffer solution, uniformly mixing, centrifuging at 6000r/min for 5min, and taking supernatant as a test sample adding standard solution.

Blank solvent: 1mL of phosphate buffer was taken as a blank solvent.

Precisely measuring 100 μ L of each of the reagent blank solution, the reference solution, the sample adding standard solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatogram, wherein the chromatogram is shown in FIG. 4, and the chromatogram of the blank solution, the reference solution, the sample adding standard solution and the sample solution is shown in FIG. 4 from top to bottom.

As can be seen from FIG. 4, the blank and other components in the sample did not interfere with the target. And the target peak and other miscellaneous peaks in the reference solution can reach the baseline separation and meet the specificity requirement.

And (3) testing a linear relation:

acceptance criteria for linear relationship: 5 points with different concentrations are prepared, the linear relation is good, and the correlation coefficient is larger than 0.990.

0.04mL, 0.10mL, 0.20mL, 0.40mL and 1.00mL of intermediate solutions of the control substances are weighed and respectively placed in 5 10mL volumetric flasks, phosphate buffer solution is added for dilution until the scales are evenly shaken, and control substance solutions of 0.2. mu.g/mL, 0.5. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL and 5.0. mu.g/mL are prepared.

Each 100. mu.L of the above control solutions was measured precisely, and injected into a chromatograph, and the linear relationship between the peak area of peak 2 and the concentration was calculated, and the results are shown in Table 4.

TABLE 4 Linear relationship of control solutions of different concentrations

The result shows that the first-order linear equation of the zymoprotein in the range of 0.2-5.0 mug/mL is as follows: y ═

109609X-20285, the correlation coefficient R is 0.9954, which is more than 0.990, and the linear relation is good.

And (3) testing the quantification limit and the detection limit:

quantification limit and detection limit acceptance criteria: the detection limit signal-to-noise ratio is greater than 3, the quantification limit signal-to-noise ratio is greater than 10, and the peak area RSD value of 6 needles for the quantification limit repeated sample introduction is less than 10%.

Precisely measuring 0.20mL of 50 mu g/mL reference substance intermediate solution, adding phosphate buffer solution to dissolve and dilute the reference substance intermediate solution to 10mL, and preparing reference substance intermediate solution a with the concentration of 1 mu g/mL; precisely measuring 0.04mL of 50 μ g/mL reference substance intermediate solution, diluting with phosphate buffer solution, diluting to 10mL, and mixing to obtain solution containing enzyme protein reference substance with concentration of 0.2 μ g/mL.

Precisely measuring 0.1mL of 1 mug/mL reference substance intermediate solution a, diluting with phosphate buffer solution, diluting to 1mL, and mixing to obtain 0.1 mug/mL reference substance solution.

100. mu.L of a control solution having a concentration of 0.2. mu.g/mL was precisely measured and injected into the chromatograph 6 times in succession, and the peak areas and the signal-to-noise ratio results are shown in Table 5.

TABLE 5 Peak area and SNR results

The result shows that the detection limit is 0.10 mug/mL, which is equivalent to 2.0mg/kg of the concentration of the test sample; the limit of quantitation is 0.20 mug/mL, which is equivalent to 4.0mg/kg of the concentration of the test sample; the peak area RSD value of 6 needles for continuous sampling with quantitative limit concentration is 5.0%.

And (3) repeatability test:

criterion for repeatability acceptability: the relative standard deviation of the results of preparing 6 test article solutions in parallel should not be more than 10%.

Preparation of a reference solution: precisely measuring a 50 mu g/mL reference substance intermediate solution in a volumetric flask with the volume of 0.1mL to 10mL, diluting the solution to a scale with a phosphate buffer solution, and shaking the solution uniformly to prepare a reference substance solution with the concentration of 0.5 mu g/mL.

Preparing a sample labeling solution: precisely weighing 0.5g of a test sample (batch number: 22051906002), adding 0.1mL of a reference substance intermediate solution of 50 mu g/mL, uniformly mixing, diluting with 6mL of phosphate buffer solution, uniformly mixing, dropwise adding 1.0mol/L of sodium hydroxide solution while shaking until the test sample is completely dissolved, fixing the volume to 10mL by using the phosphate buffer solution, uniformly mixing, centrifuging at 6000r/min for 5min, and taking the supernatant as a test sample standard solution.

Precisely measuring 100 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, recording chromatogram, and calculating the content RSD% of the 6 sample solutions prepared in parallel, the results are shown in Table 6.

TABLE 6 repeatability tests

Since no enzyme protein was detected in the test sample, the reproducibility was tested by quantitatively adding a control to the test sample. The results show that the content RSD of the solution of 6 samples prepared in parallel is 1.5 percent and less than 10 percent, and the repeatability of the method is good.

And (3) testing intermediate precision:

intermediate precision acceptance criteria: the relative standard deviation of the results of 12 test solutions prepared in parallel by two experimenters is not more than 15%.

At different times, 6 parts of the test sample (batch number: 22051906002) were precisely weighed by another laboratory, a penicillin G acylase control was quantitatively added to the test sample, and the test was carried out on a repetitive basis, and the content was measured by HPLC. The results of the measurements on the two test solutions processed by the two laboratory workers are shown in Table 7.

TABLE 7 intermediate precision test

Because no enzyme protein is detected in the test sample, the intermediate precision is tested by quantitatively adding a reference substance into the test sample, the result RSD of 12 test samples prepared by two analysts in parallel is 3.0 percent and less than 15 percent, and the intermediate precision of the method is good.

And (3) testing accuracy:

acceptable standard of accuracy: the recovery rate of each concentration is 80-120%, and the Relative Standard Deviation (RSD) of the recovery rate is not more than 10%.

Control solution: accurately weighing 0.05mL, 0.10mL and 0.15mL of 50 μ g/mL control intermediate solution, respectively adding into three 10mL volumetric flasks, diluting to scale with phosphate buffer solution, shaking to obtain control solutions with concentrations of 0.25 μ g/mL, 0.5 μ g/mL and 0.75 μ g/mL.

Preparing a sample labeling solution: precisely weighing three groups of test samples, wherein each group comprises three parts, each part comprises 0.5g of cefaclor raw material, respectively adding 0.05mL, 0.10mL and 0.15mL of intermediate solution of a reference substance into the three groups of test samples, uniformly mixing, diluting with a phosphate buffer solution, uniformly mixing, dropwise adding 1.0mol/L sodium hydroxide solution, oscillating while dropwise adding until the test samples are completely dissolved, diluting to 10mL with the phosphate buffer solution, uniformly mixing, centrifuging at 6000r/min for 5min, and taking supernatant as a test sample standard solution.

Precisely measuring 100 mu L of reference solution and sample standard solution, injecting into chromatograph, recording chromatogram, and calculating RSD content of sample standard solution of each concentration. The results are shown in Table 8.

TABLE 8 accuracy test

The standard recovery rates of the three levels are 97.9%, 105.7% and 96.5% respectively, the requirement that the recovery rates are all between 80% and 120% is met, and the RSD values are 8.0%, 0.8% and 6.8% and are all less than 10%, which indicates that the accuracy of the method is good.

And (3) stability testing:

acceptable standards for solution stability: the Relative Standard Deviation (RSD) of the peak areas at different times should be no more than 10%.

Precisely measuring a 50 mu g/mL reference substance intermediate solution in a volumetric flask with the volume of 0.15mL to 10mL, diluting the volumetric flask to a scale with a phosphate buffer solution, and shaking up to prepare a reference substance solution with the concentration of 0.75 mu g/mL.

Measuring the reference solution for 0h, 5h, 8h, 12h, 16h, 20h and 24h respectively to obtain 100 μ L, injecting into chromatograph, recording chromatogram, calculating RSD% of peak area of peak 2, and examining solution stability. The results are shown in Table 9.

TABLE 9 control solution stability

Time (h)	Peak area of control solution
		0	69784
5	72288
		8	73208
12	60964
		16	60119
20	61892
		24	55068
20 hours RSD (%)	9.1

The result shows that the control solution is placed at room temperature for 20 hours, the peak area RSD is 9.1 percent and is less than 10 percent, and the solution stability is good.

Precisely weighing 0.5g of a test sample (batch number: 22051906002), precisely weighing 0.15mL of a reference sample intermediate solution with the concentration of 50 mu g/mL, adding the intermediate solution into the test sample, uniformly mixing, diluting with 6mL of phosphate buffer solution, uniformly mixing, dropwise adding 1.0mol/L sodium hydroxide solution while shaking until the test sample is completely dissolved, fixing the volume to 10mL with the phosphate buffer solution, uniformly mixing, centrifuging at 6000r/min for 5min, and taking a supernatant as a test sample adding standard solution.

Measuring the sample and adding 100 μ L of standard solution at 0h, 5h, 11h, 15h, 19h and 24h respectively, injecting into chromatograph, recording chromatogram, calculating RSD% of peak area of peak 2, and examining solution stability. The results are shown in Table 10.

TABLE 10 stability of the test sample spiking solutions

The result shows that the sample added with the standard solution is placed for 24 hours at room temperature, the peak area RSD is 6.2 percent and is less than 10 percent, and the solution stability is good.

And (3) durability test:

acceptable criteria for durability: the separation degree of the target peak from other peaks is more than 1.5; the peak area results show RSD values of less than 10%.

Precisely weighing 0.5g of a test sample (batch number: 22051906002), placing the test sample into a 10mL volumetric flask, precisely weighing 0.15mL of a reference sample intermediate solution of 50 mu g/mL, adding the test sample, uniformly mixing, adding 6mL of phosphate buffer solution, uniformly mixing, dropwise adding 1.0mol/L sodium hydroxide solution, shaking while dropwise adding until the test sample is dissolved, adding the phosphate buffer solution to dilute to a scale, shaking uniformly, centrifuging at 6000r/min for 5min, and taking the supernatant as a test sample standard solution.

Precisely measuring 100 mu L of each of the reference solution and the sample standard solution under the following conditions: the column temperature is +/-2 ℃, the flow rate is +/-0.1 mL/min, the organic phase ratio is +/-1%, the mixture is injected into a liquid chromatograph, a chromatogram is recorded, and the system applicability is considered, wherein the system applicability is calculated by the peak area of the peak 2. The results are shown in Table 11.

TABLE 11 durability test

The result shows that the organic phase ratio is +/-1%, the column temperature is +/-2 ℃, the flow rate is +/-0.1 mL/min, the separation degree of the target peak and the impurity peak of the sample added with the standard solution under the condition of changing the chromatographic conditions is more than 1.5, the tailing factor is between 0.90 and 1.06, the peak area RSD value is less than 10 percent, and the method has good durability.

Example 4

Measuring appropriate amount of reference substance, diluting with phosphate buffer solution, and mixing to obtain reference substance solution with concentration of 0.5 μ g/mL.

Precisely weighing 0.5g of samples of different batches, diluting the samples by 6mL of phosphate buffer solution, dropwise adding 1.0mol/L of sodium hydroxide solution, oscillating while dropwise adding until the samples are completely dissolved, fixing the volume to 10mL by using the phosphate buffer solution, uniformly mixing, centrifuging at 6000r/min for 5min, and taking supernatant as the sample solution.

The control solution and the sample solution were taken at 100. mu.L each and injected into a liquid chromatograph, and the mobile phase liquid phase gradient is shown in Table 12. The detection is carried out by a fluorescence detector, a chromatogram is recorded, an external standard method is adopted according to the peak area, and the detection result is shown in Table 13.

TABLE 12 liquid phase gradient conditions

TABLE 13 cefaclor raw material test results

Note: the detection limit in Table 13 was 2.0 mg/kg.

The method adopts liquid chromatography to determine the residual quantity of penicillin G acylase in cefaclor raw materials, takes the penicillin G acylase as an enzyme protein standard, uses a chromatographic column TSK gel octadecenyl-4 PW with good protein separation effect to separate, uses a liquid chromatography-fluorescence detector to detect, and obtains PGA which presents good first-order linear relation in the range of 0.2-5.0 mu G/mL and is quantified by an external standard method. The detection limit of the method is 2.0mg/kg, the quantification limit is 4.0mg/kg, and the method is simple and convenient and has strong operability.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting the content of penicillin G acylase in cefaclor is characterized by comprising the following steps:

2. The detection method according to claim 1, wherein the phosphate buffer solution in steps 1) to 4) is prepared from monopotassium phosphate, a sodium hydroxide solution and water, the mass-to-volume ratio of the monopotassium phosphate to the sodium hydroxide solution to the water is 6-7.5 g: 25-35 mL: 950-1000 mL, and the concentration of the sodium hydroxide solution is 0.8-1.2 mol/L.

3. The detection method according to claim 1 or 2, wherein the concentration of penicillin G acylase in the control intermediate solution of step 1) is 45-55 μ G/mL; in the penicillin G acylase reference substance, the total protein content is 11-13 mg/mL.

4. The assay as claimed in claim 3 wherein the gradient concentrations of penicillin G acylase in step 2) of the control solution are 0.1 μ G/mL, 0.2 μ G/mL, 0.5 μ G/mL, 1.0 μ G/mL, 2.0 μ G/mL and 5.0 μ G/mL, respectively.

5. The detection method according to claim 4, wherein the mass-to-volume ratio of the test cefaclor, the phosphate buffer solution and the sodium hydroxide solution in the step 3) is 0.4-0.6 g: 8-10 mL: 0.5-3 mL;

6. The detection method according to claim 4 or 5, wherein in the steps 3) and 4), the concentration of the sodium hydroxide solution is 0.8-1.2 mol/L, the rotation speed of the centrifugal treatment is 5500-6500 r/min, and the time is 3-8 min.

7. The detection method according to claim 6, wherein in the detection process of the step 5) and the step 6), gradient elution is performed by using a mixed solution of a mobile phase A and a mobile phase B; in the mixed solution, the volume ratio of the mobile phase A to the mobile phase B is 35-60: 40-65; the time of gradient elution is less than or equal to 1 h.

8. The detection method according to claim 7, wherein the mobile phase A and the mobile phase B each comprise water, acetonitrile, and trifluoroacetic acid; in the mobile phase A, the volume ratio of water to acetonitrile to trifluoroacetic acid is 940-960: 45-55: 4-6, and in the mobile phase B, the volume ratio of water to acetonitrile to trifluoroacetic acid is 95-105: 880-920: 4-6.

9. The detection method according to claim 7 or 8, wherein in the detection process of the step 5) and the step 6), the flow rate is 0.8-1.2 mL/min, the column temperature is 35-45 ℃, and the sample injection amount is 50-100 μ L; the excitation wavelength is 270-280 nm, and the emission wavelength is 340-350 nm.