Detection Method of Imidazole Chloride by High Performance Liquid Chromatography

Abstract

The invention belongs to the field of pharmaceutical detection technology, and is specifically related to a high performance liquid chromatography detection method for imidazole chloride. The detection method adopts high performance liquid chromatography, adopts C18 chromatographic column, detection wavelength 200-220nm, column room temperature, acetonitrile-10mol / L potassium hydrogen phthalate buffer as mobile phase, flow rate 0.5-1.2ml / min, isocratic elution; Derivatization treatment is carried out before sample detection, using amine compounds as derivatization reagents, using alkaline compounds as catalysts, using anhydrous ethanol as solvent, reacting 5-20min at room temperature, completing derivatization treatment. The detection method of the present invention is convenient and concise, highly sensitive, good chromatographic peak shape, accurate and reliable data, good reproducibility, can accurately detect the content of imidazole chloride, improve the safety of medicines, and effectively control the quality of medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a high performance liquid chromatography detection method for imidazole chloride. Background Art

[0002] (1-Chlorocarbonyl-2-Imidazolidone), referred to as imidazole chloride, has the following structural formula:

[0003]

[0004] 1-Chloroformyl-2-imidazolidinone is an important intermediate for the semi-synthetic penicillin mezlocillin sodium and azlocillin sodium, and is widely used in the synthesis of anti-schistosomiasis and chemical sterilization drugs, insecticides and insecticides. This drug is currently a commonly used anti-infective drug in clinical practice. It has been used in more than 20 countries including my country and is included in the United States Pharmacopoeia (USPXXⅢ) as a basic drug in my country. With the widespread application of mezlocillin sodium and azlocillin sodium, imidazole chloride, as an important intermediate for the synthesis of mezlocillin sodium and azlocillin sodium, has a good market prospect.

[0005] The detection methods for imidazole chloride reported so far mainly include the volumetric analysis method of silver volumetric method, and the detection of imidazole chloride by titration method. The determination of the titration end point is based on the color change, which has a large observation error. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a high performance liquid chromatography detection method for imidazole chloride, which is convenient and concise, has high sensitivity, good chromatographic peak shape, accurate and reliable data, good reproducibility, can accurately detect the content of imidazole chloride, improve the safety of drugs, and effectively control the quality of drugs.

[0007] The high performance liquid chromatography detection method of imidazole chloride of the present invention adopts high performance liquid chromatography, adopts C18 chromatographic column, detection wavelength is 200-220nm, column room temperature, acetonitrile-10mol / L potassium hydrogen phthalate buffer as mobile phase, flow rate is 0.5-1.2ml / min, isocratic elution;

[0008] The samples were subjected to derivatization treatment before testing, using an amine compound as a derivatization reagent, an alkaline compound as a catalyst, and anhydrous ethanol as a solvent, and reacted at room temperature for 5-20 minutes to complete the derivatization treatment.

[0009] Preferably, the chromatographic column has a specification of 250 mm×4.6 mm and 5 μm.

[0010] Preferably, the detection wavelength is 210 nm.

[0011] Preferably, the volume ratio of acetonitrile to 10 mol / L potassium hydrogen phthalate buffer in the mobile phase is (70-80):(30-20), and more preferably 70:30.

[0012] Preferably, the flow rate is 0.8 ml / min.

[0013] Preferably, the injection volume is 20uL.

[0014] Preferably, the elution time is 20 min.

[0015] Preferably, the detection method comprises the following steps:

[0016] (1) Sample derivatization treatment;

[0017] (2) preparing a control sample solution and a test sample solution of imidazole chloride respectively;

[0018] (3) Prepare a standard curve;

[0019] (4) Calculate the content of the test sample.

[0020] Preferably, the derivatization agent is one or more of methylamine, aniline, ethylenediamine, and diethylamine, and aniline is most preferred.

[0021] Preferably, the catalyst is one or more of sodium hydroxide, sodium carbonate, pyridine, and triethylamine, and most preferably sodium carbonate.

[0022] Through experiments, under the best liquid chromatography conditions, the content of imidazole chloride has a good linear relationship in the concentration range of 0.5-1.5 mg / ml, and the correlation coefficient is above 0.99. The blank matrix matching standard solution method is used for standard calibration, showing that the average recovery rate of imidazole chloride is above 93%, and the relative standard deviation is less than 2.0%. This method is suitable for detecting the content of imidazole chloride.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention adopts a derivatization method, first reacting imidazole chloride with amine to form a stable amide, and then performing high performance liquid chromatography analysis to avoid the error caused by the titration method;

[0025] (2) The detection method of the present invention is convenient and concise, has high sensitivity, good chromatographic peak shape, accurate and reliable data, good reproducibility, can accurately detect the content of imidazole chloride, improve the safety of drugs, effectively control the quality of drugs, and play a guiding role in the use of downstream customers. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the HPLC chromatogram of the standard product in Example 1 of the present invention;

[0027] Figure 2 It is a standard curve diagram of the concentration-peak area of ​​imidazole chloride in Example 2 of the present invention;

[0028] Figure 3 It is the high performance liquid chromatogram of imidazole chloride sample 1 in Example 4 of the present invention;

[0029] Figure 4 It is the HPLC chromatogram of imidazole chloride sample 2 in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto, and any changes made to the technical solution of the present invention by professionals in the field should all fall within the protection scope of the present invention.

[0031] The raw materials used in the examples, unless otherwise specified, are all commercially available conventional raw materials; the process methods used in the examples, unless otherwise specified, are all conventional methods in the art.

[0032] In the following examples, the HPLC conditions were as follows:

[0033] Chromatographic column: C18, 250×4.6mm, 5μm;

[0034] Column temperature: room temperature;

[0035] Detection wavelength: 210nm;

[0036] Mobile phase: acetonitrile-10 mol / L potassium hydrogen phthalate buffer with a volume ratio of 70:30;

[0037] Flow rate: 0.8ml / min;

[0038] Injection volume: 20 μl;

[0039] Elution time: 20min.

[0040] (1) Selection of mobile phase ratio:

[0041] The effects of different volume ratios of acetonitrile-10 mol / L potassium hydrogen phthalate buffer on the separation of the test substances were investigated. The results are shown in Table 1.

[0042] Table 1

[0043] Volume ratio of acetonitrile-10mol / L potassium hydrogen phthalate buffer Imidazole chloride retention time (min) 80:20 1.295 75:25 2.134 70:30 3.707 65:35 6.053 60:40 7.254

[0044] From the results in Table 1, it can be seen that when the volume ratio of acetonitrile in the mobile phase changes from 80-60%, the retention time gradually increases as the concentration decreases. However, when the acetonitrile concentration is lower than 70%, the separation conditions do not reach the optimal state, and the impurities and imidazole chloride are not completely separated. Therefore, the optimal volume ratio of acetonitrile-10mol / L potassium hydrogen phthalate buffer is 70:30.

[0045] (2) Selection of detection wavelength:

[0046] By examining the ultraviolet absorption intensity of the imidazole chloride derivatization product at different wavelengths, the results show that the ultraviolet absorption intensity of the imidazole chloride derivatization product is stronger when the ultraviolet absorption wavelength is 200-220nm, so 210nm is preferably used as its detection wavelength.

[0047] (3) Selection of flow rate:

[0048] The effect of different flow rates on the separation of the analytes was investigated. The results showed that when the flow rate changes from 0.5-1.2 ml / min, the retention time will be very long when the flow rate is lower than 0.5 ml / min, and the separation effect of the analytes will be affected when the flow rate is higher than 1.2 ml / min, and the impurities and imidazole chloride are not completely separated. Therefore, the preferred flow rate is 0.8 ml / min.

[0049] Examples 1-3 respectively test the system applicability, standard curve and detection limit, recovery rate and precision, and actual sample determination of the detection method, wherein the preparation method of the sample solution to be tested is as follows:

[0050] (1) Preparation of reference solution: accurately weigh 25.0 mg of imidazole chloride reference substance and place it in a 25 ml volumetric flask, add 10 ml of anhydrous ethanol, 0.02 ml of aniline and 0.5 mg of sodium carbonate, shake, let stand for 20 min, then make up to volume with ethanol, shake well, and let stand to prepare the reference solution;

[0051] (2) Preparation of test solution: Accurately weigh 25.0 mg of the test sample and place it in a 25 ml volumetric flask. Add 10 ml of anhydrous ethanol, 0.02 ml of aniline and 0.5 mg of sodium carbonate. Shake and let stand for 20 min. Then make up to volume with ethanol, shake well and let stand to prepare the test solution.

[0052] Example 1

[0053] System suitability experiment:

[0054] Accurately weigh 50.0 mg of imidazole chloride standard, place it in a 50 ml volumetric flask, add 20 ml of anhydrous ethanol, 0.04 ml of aniline and 1.0 mg of sodium carbonate, shake, let stand for 20 min, then make up to volume with ethanol, shake well, and use this as the system suitability solution. Take 20 μl of the system suitability solution and inject it into the liquid chromatograph for testing. Record the chromatogram, such as Figure 1 shown.

[0055] Depend on Figure 1 It can be seen that the peak shape is good, the solvent has no effect on the main peak and other impurity peaks, and the retention time of the imidazole chloride standard under the preferred chromatographic conditions is basically consistent with the retention time of the imidazole chloride test sample. Therefore, the preferred chromatographic conditions are suitable for the detection of imidazole chloride.

[0056] Example 2

[0057] Standard curve and detection limit experiment:

[0058] Accurately weigh 50.0 mg of imidazole chloride standard, place it in a 50 ml volumetric flask, add 20 ml of anhydrous ethanol, 0.04 ml of aniline and 1.0 mg of sodium carbonate, shake, let stand for 20 minutes, then make up to volume with ethanol, shake well, and prepare a standard solution with a concentration of 1 mg / ml; accurately weigh 2 ml, 4 ml, 6 ml, 8 ml and 10 ml of the above standard solution respectively, place it in a 10 ml volumetric flask, make up to 10 ml with mobile phase, and prepare imidazole chloride standard solutions with concentrations of 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml and 1.0 mg / ml. Inject and measure under the preferred chromatographic conditions. Each concentration is measured 5 times in parallel, and a standard curve is made for the imidazole chloride concentration based on the average value of the peak area obtained to obtain the normalized equation and correlation coefficient diagram, as shown in the figure. Figure 2 shown.

[0059] Depend on Figure 2 It can be seen that the linear condition is good.

[0060] Example 3

[0061] Recovery and precision experiments:

[0062] The blank spike method was used to determine the recovery and precision. Three concentrations of 0.2, 0.6, and 1.0 mg / ml of standard products were added to the blank sample, and each concentration sample was measured 5 times. The peak area was recorded and entered into the regression equation, and the recovery and RSD were calculated according to the formula. The results are shown in Table 2.

[0063] Table 2

[0064] Standard addition amount (mg / ml) Sample measurement value (mg / ml) Recovery rate (%) 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

[0065] It can be seen from Table 2 that the recovery rate of the detection method is above 90%, and the RSD of the peak area of ​​imidazole chloride is less than 2.0%, which shows that the recovery rate and system precision of the detection method are good.

[0066] Example 4

[0067] Sample determination:

[0068] (1) The imidazole chloride sample 1 was measured according to the preferred HPLC conditions. The spectrum is as follows Figure 3 shown.

[0069] from Figure 3 It can be seen that the purity of imidazole chloride is 86.3092%, and the content is calculated to be 81.79%

[0070] (2) The imidazole chloride sample 2 was measured according to the preferred HPLC conditions. The spectrum is as follows Figure 4 shown.

[0071] from Figure 4 It can be seen that the purity of imidazole chloride is 86.7654%, and the content is calculated to be 84.66%.

Claims

1. A high performance liquid chromatography detection method for imidazole chloride, characterized in that: The method comprises the following steps: adopting a high performance liquid chromatography method, using a C18 chromatographic column, a detection wavelength of 200-220 nm, a column room temperature, using acetonitrile-10 mol / L potassium hydrogen phthalate buffer as the mobile phase, a flow rate of 0.5-1.2 ml / min, and isocratic elution; the volume ratio of acetonitrile to 10 mol / L potassium hydrogen phthalate buffer in the mobile phase is (70-80):(30-20); The sample is subjected to derivatization treatment before detection, using an amine compound as a derivatization reagent, an alkaline compound as a catalyst, and anhydrous ethanol as a solvent, reacting at room temperature for 5-20 minutes to complete the derivatization treatment; The derivatization reagent is aniline; The catalyst is sodium carbonate.

2. The high performance liquid chromatography detection method of imidazole chloride according to claim 1, wherein: The specifications of the chromatographic column are 250mm×4.6mm, 5μm.

3. The high performance liquid chromatography detection method of imidazole chloride according to claim 1, wherein: The detection wavelength is 210 nm.

4. The high performance liquid chromatography detection method of imidazole chloride according to claim 1, wherein: The flow rate is 0.8 ml / min.

5. The high performance liquid chromatography detection method of imidazole chloride according to claim 1, wherein: The injection volume was 20uL.

6. The high performance liquid chromatography detection method of imidazole chloride according to claim 1, wherein: The elution time is 20 min.

7. The high performance liquid chromatography detection method of imidazole chloride according to claim 1, wherein: The detection method comprises the following steps: (1) Sample derivatization treatment; (2) preparing a control sample solution and a test sample solution of imidazole chloride respectively; (3) Prepare a standard curve; (4) Calculate the content of the test sample.

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