CN116223650A - High performance liquid chromatography detection method of imidazole acyl chloride - Google Patents

High performance liquid chromatography detection method of imidazole acyl chloride Download PDF

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CN116223650A
CN116223650A CN202211614578.7A CN202211614578A CN116223650A CN 116223650 A CN116223650 A CN 116223650A CN 202211614578 A CN202211614578 A CN 202211614578A CN 116223650 A CN116223650 A CN 116223650A
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performance liquid
high performance
liquid chromatography
imidazole
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孟祥安
常明珠
张立明
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Shandong Xinquan Pharmaceutical Co ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

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Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a high performance liquid chromatography detection method of imidazole acyl chloride. The detection method adopts high performance liquid chromatography, adopts a C18 chromatographic column, and adopts a detection wavelength of 200-220nm, a column Wen Shiwen, adopts acetonitrile-10 mol/L potassium hydrogen phthalate buffer solution as a mobile phase, and has a flow rate of 0.5-1.2ml/min, and isocratic elution; derivatization treatment is carried out before sample detection, an amine compound is used as a derivatization reagent, an alkaline compound is used as a catalyst, absolute ethyl alcohol is used as a solvent, and the derivatization treatment is completed after the reaction is carried out for 5 to 20 minutes at room temperature. The detection method disclosed by the invention is convenient and concise, high in sensitivity, good in chromatographic peak shape, accurate and reliable in data, good in reproducibility, capable of accurately detecting the content of the imidazole acyl chloride, improving the safety of medicines and effectively controlling the quality of medicines.

Description

High performance liquid chromatography detection method of imidazole acyl chloride
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a high performance liquid chromatography detection method of imidazole acyl chloride.
Background
(1-Chlorocarbonyl-2-Imidazolidone), namely imidazole chloride, has the following structural formula:
Figure BDA0003996326180000011
the 1-chloroformyl-2-imidazolidinone is an important intermediate of semisynthetic penicillin mezlocillin sodium and azlocillin sodium, is widely used for synthesizing anti-schistosomiasis and chemical sterilization drugs, pesticides and the like, is an anti-infective drug commonly used clinically at present, has been applied to more than 20 countries including China to date, and is received in United States Pharmacopoeia (USPXXIII) as a basic drug of China. With the wide application of mezlocillin sodium and azlocillin sodium, the imidazole acyl chloride is used as an important intermediate for synthesizing mezlocillin sodium and azlocillin sodium, and has good market prospect.
The currently reported detection method of the imidazole acyl chloride mainly comprises a silver amount method of a capacity analysis method, the imidazole acyl chloride is detected by a titration method, and the judgment of a titration end point is based on color change and has a large observation error.
Disclosure of Invention
The invention aims to solve the technical problems that: the high-performance liquid chromatography detection method for the imidazole acyl chloride is convenient, simple, high in sensitivity, good in chromatographic peak shape, accurate and reliable in data, good in reproducibility, capable of accurately detecting the content of the imidazole acyl chloride, improving the safety of medicines and effectively controlling the quality of the medicines.
The high performance liquid chromatography detection method of the imidazole acyl chloride adopts a high performance liquid chromatography method, adopts a C18 chromatographic column, and adopts a detection wavelength of 200-220nm and a column Wen Shiwen, and adopts acetonitrile-10 mol/L potassium hydrogen phthalate buffer solution as a mobile phase, the flow rate is 0.5-1.2ml/min, and the elution is isocratic;
derivatization treatment is carried out before sample detection, an amine compound is used as a derivatization reagent, an alkaline compound is used as a catalyst, absolute ethyl alcohol is used as a solvent, and the derivatization treatment is completed after the reaction is carried out for 5 to 20 minutes at room temperature.
Preferably, the column format is 250mm by 4.6mm,5 μm.
Preferably, the detection wavelength is 210nm.
Preferably, the volume ratio of acetonitrile to 10mol/L potassium hydrogen phthalate buffer in the mobile phase is (70-80): (30-20), more preferably 70:30.
Preferably, the flow rate is 0.8ml/min.
Preferably, the sample volume is 20uL.
Preferably, the elution time is 20min.
Preferably, the detection method comprises the following steps:
(1) Sample derivatization treatment;
(2) Respectively preparing a control sample solution and a test sample solution of imidazole chloride;
(3) Manufacturing a standard curve;
(4) And calculating the content of the test sample.
Preferably, the derivatizing agent is one or more of methylamine, aniline, ethylenediamine, diethylamine, most preferably aniline.
Preferably, the catalyst is one or more of sodium hydroxide, sodium carbonate, pyridine and triethylamine, and most preferably sodium carbonate.
Through experiments, under the optimal liquid chromatography condition, the content of the imidazole acyl chloride has good linear relation within the concentration range of 0.5-1.5 mg/ml, the correlation coefficient is more than 0.99, and standard correction is carried out by adopting a blank matrix matching standard solution method, so that the average recovery rate of the imidazole acyl chloride is more than 93%, and the relative standard deviation is less than 2.0%. The method is suitable for detecting the content of the imidazole acyl chloride.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention adopts a derivatization method, firstly, imidazole acyl chloride and amine react to generate stable amide, and then high performance liquid chromatography analysis is carried out, so that errors caused by a titration method are avoided;
(2) The detection method disclosed by the invention is convenient and concise, high in sensitivity, good in chromatographic peak shape, accurate and reliable in data, good in reproducibility, capable of accurately detecting the content of the imidazole acyl chloride, improving the safety of medicines, effectively controlling the quality of medicines and playing a guiding role for downstream customers.
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FIG. 1 is a high performance liquid chromatogram of the standard of example 1 of the present invention;
FIG. 2 is a standard graph of concentration versus peak area for imidazole chloride in example 2 of the present invention;
FIG. 3 is a high performance liquid chromatogram of the imidazole chloride test sample 1 in example 4 of the present invention;
FIG. 4 is a high performance liquid chromatogram of imidazole chloride test sample 2 in example 4 of the present invention.
Detailed Description
The present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto, and modifications made by those skilled in the art to the technical scheme of the present invention shall fall within the scope of the present invention.
The raw materials used in the examples, unless otherwise specified, were all commercially available conventional raw materials; the process used in the examples, unless otherwise specified, is conventional in the art.
In the following examples, the conditions of high performance liquid chromatography are as follows:
chromatographic column: c18 250X 4.6mm,5 μm;
column temperature: room temperature;
detection wavelength: 210nm;
mobile phase: acetonitrile-10 mol/L potassium hydrogen phthalate buffer solution with the volume ratio of 70:30;
flow rate: 0.8ml/min;
sample injection amount: 20 μl;
elution time: 20min.
(1) Selection of mobile phase proportions:
the effect of acetonitrile-10 mol/L potassium hydrogen phthalate buffer solution on the separation effect of substances to be tested was examined, and the results are shown in Table 1.
TABLE 1
Volume ratio of acetonitrile-10 mol/L potassium hydrogen phthalate buffer solution Imidazole acyl chloride retention time (min)
80:20 1.295
75:25 2.134
70:30 3.707
65:35 6.053
60:40 7.254
As can be seen from the results of Table 1, the retention time was gradually prolonged as the concentration was decreased in the course of the change of the acetonitrile volume ratio in the mobile phase from 80 to 60%, but when the acetonitrile concentration was lower than 70%, the separation condition was not reached to the optimum state, and the impurity and the imidazole chloride were not completely separated, so that the optimum volume ratio of acetonitrile-10 mol/L potassium hydrogen phthalate buffer was 70:30.
(2) Selection of detection wavelength:
by examining the ultraviolet absorption intensity of the imidazole acyl chloride derivative products at different wavelengths. The results show that the ultraviolet absorption intensity of the imidazole chloride derivative product is strong when the ultraviolet absorption wavelength is in the range of 200-220nm, and thus 210nm is preferable as its detection wavelength.
(3) Selection of flow rates:
by examining the influence of different flow rates on the separation effect of the object to be detected. The results show that the retention time is long when the flow rate is lower than 0.5ml/min in the process of changing the flow rate from 0.5ml/min to 1.2ml/min, and the separation effect of the object to be detected is affected when the flow rate is higher than 1.2ml/min, and impurities and imidazole chloride are not completely separated. Therefore, the flow rate is preferably 0.8ml/min.
Examples 1-3 were tested for system applicability, standard curve and limit of detection, recovery and precision, and actual sample determination, respectively, wherein the preparation method of the sample solution to be tested is as follows:
(1) Preparing a reference substance solution: precisely weighing imidazole chloride reference substance 25.0mg, placing in a 25ml volumetric flask, adding 10ml absolute ethanol, 0.02ml aniline and 0.5mg sodium carbonate, oscillating, standing for 20min, fixing volume with ethanol, shaking, standing to obtain reference substance solution;
(2) Preparing a test solution: precisely weighing 25.0mg of the sample, placing in a 25ml volumetric flask, adding 10ml of absolute ethyl alcohol, 0.02ml of aniline and 0.5mg of sodium carbonate, oscillating, standing for 20min, fixing the volume with ethanol, shaking, and standing to obtain the sample solution.
Example 1
System applicability experiment:
precisely weighing 50.0mg of imidazole chloride standard substance, placing in a 50ml volumetric flask, adding 20ml of absolute ethyl alcohol, 0.04ml of aniline and 1.0mg of sodium carbonate, oscillating, placing for 20min, fixing the volume with ethanol, shaking uniformly to obtain a system applicability solution, taking 20 μl of the system applicability solution, injecting into a liquid chromatograph, testing, and recording a chromatogram, as shown in figure 1.
As can be seen from FIG. 1, the peak pattern is good, the solvent has no influence on the main peak and other impurity peaks, and the retention time of the imidazole acyl chloride standard substance under the preferable chromatographic condition is basically consistent with that of the imidazole acyl chloride test substance, so that the preferable chromatographic condition is suitable for detecting the imidazole acyl chloride.
Example 2
Standard curve and limit of detection experiments:
precisely weighing 50.0mg of imidazole chloride standard substance, placing in a 50ml volumetric flask, adding 20ml of absolute ethyl alcohol, 0.04ml of aniline and 1.0mg of sodium carbonate, oscillating, standing for 20min, then fixing the volume with ethanol, and shaking uniformly to prepare a standard solution with the concentration of 1 mg/ml; 2ml, 4ml, 6ml, 8ml and 10ml of the standard solution are respectively weighed precisely and placed in a volumetric flask of 10ml, and the mobile phase is used for fixing the volume to 10ml to prepare the imidazolyl chloride standard solutions with the concentration of 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1.0 mg/ml. The assay is performed under preferred chromatographic conditions. Each concentration was measured 5 times in parallel, and the imidazole chloride concentration was subjected to standard curve according to the obtained peak area average value, so as to obtain a graph of the normalization equation and the correlation coefficient, as shown in fig. 2.
As can be seen from fig. 2, the linearity condition is good.
Example 3
Recovery rate and precision experiment:
and (5) adopting a blank labeling method to measure the recovery rate and the precision. Standard substances of 0.2, 0.6 and 1.0mg/ml of 3 concentrations are added into blank samples, each concentration sample is measured 5 times, peak areas are recorded and respectively brought into a regression equation, and recovery rate and RSD are respectively calculated according to the formulas. The results are shown in Table 2.
TABLE 2
Standard addition (mg/ml) Sample measurement value (mg/ml) Recovery (%) RSD(％)
0.2 0.180 90.0 1.527
0.6 0.571 95.2 1.038
1.0 0.942 94.2 1.247
As can be seen from Table 2, the recovery rate of the detection method is more than 90%, the RSD of the peak area of the imidazole acyl chloride is less than 2.0%, and the recovery rate and the system precision of the detection method are good.
Example 4
Sample measurement:
(1) The imidazole chloride sample 1 was measured according to the preferred high performance liquid chromatography conditions, and the spectrum is shown in fig. 3.
As can be seen from FIG. 3, the purity of the imidazole acyl chloride is 86.3092%, and the content is 81.79% calculated
(2) The imidazole chloride sample 2 was measured according to the preferred high performance liquid chromatography conditions, and the spectrum is shown in fig. 4.
As can be seen from FIG. 4, the purity of the imidazole acyl chloride was 86.7654%, and the content was calculated to be 84.66%.

Claims (10)

1. A high performance liquid chromatography detection method of imidazole acyl chloride is characterized in that: adopting high performance liquid chromatography, adopting C18 chromatographic column, detecting wavelength of 200-220nm, column Wen Shiwen, using acetonitrile-10 mol/L potassium hydrogen phthalate buffer solution as mobile phase, and eluting at flow rate of 0.5-1.2ml/min at equal degree;
derivatization treatment is carried out before sample detection, an amine compound is used as a derivatization reagent, an alkaline compound is used as a catalyst, absolute ethyl alcohol is used as a solvent, and the derivatization treatment is completed after the reaction is carried out for 5 to 20 minutes at room temperature.
2. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the column size was 250mm by 4.6mm,5 μm.
3. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the detection wavelength was 210nm.
4. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the volume ratio of acetonitrile to 10mol/L potassium hydrogen phthalate buffer solution in the mobile phase is (70-80): 30-20.
5. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the flow rate was 0.8ml/min.
6. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the sample volume was 20uL.
7. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the elution time was 20min.
8. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the detection method comprises the following steps:
(1) Sample derivatization treatment;
(2) Respectively preparing a control sample solution and a test sample solution of imidazole chloride;
(3) Manufacturing a standard curve;
(4) And calculating the content of the test sample.
9. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the derivatization reagent is one or more of methylamine, aniline, ethylenediamine and diethylamine.
10. The method for detecting the imidazole chloride by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: the catalyst is one or more of sodium hydroxide, sodium carbonate, pyridine and triethylamine.
CN202211614578.7A 2022-12-13 2022-12-13 High performance liquid chromatography detection method of imidazole acyl chloride Pending CN116223650A (en)

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