CN117825547A - Isolation assay of clotrimazole intermediate Z 1 High performance liquid chromatography of related substances in (B) - Google Patents

Isolation assay of clotrimazole intermediate Z 1 High performance liquid chromatography of related substances in (B) Download PDF

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CN117825547A
CN117825547A CN202311776084.3A CN202311776084A CN117825547A CN 117825547 A CN117825547 A CN 117825547A CN 202311776084 A CN202311776084 A CN 202311776084A CN 117825547 A CN117825547 A CN 117825547A
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impurity
mobile phase
formula
clotrimazole
volume ratio
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黄诗婕
王绍丽
陈雯
周书荣
唐舒棠
刘阔
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Chongqing Huapont Pharm Co Ltd
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    • 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
    • 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
    • G01N30/06—Preparation
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • 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
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    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • 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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Abstract

The invention belongs to the technical field of pharmaceutical chemistry analysis, and particularly relates to a method for separating and measuring a clotrimazole intermediate Z 1 High performance liquid chromatography of the related substances. The high performance liquid chromatography adopts octadecylsilane chemically bonded silica as a chromatographic column filler, phosphoric acid aqueous solution as a mobile phase A and methanol as a mobile phase B, and the clotrimazole intermediate Z is eluted by a linear gradient 1 Separating related substances in the water; detecting by a detector with detection wavelength of 220-230nm to obtain a chromatogram; and then according to the chromatogram, calculating the content of each impurity by adopting an external standard method and/or a main component self-contrast method. The method has the advantages of simple operation, high specificity, high sensitivity and good durabilityHigh accuracy, and successfully realizes the clotrimazole intermediate Z 1 Qualitative and quantitative detection of various impurities in the sample.

Description

Isolation assay of clotrimazole intermediate Z 1 High performance liquid chromatography of related substances in (B)
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry analysis, and particularly relates to a method for separating and measuring a clotrimazole intermediate Z 1 High performance liquid chromatography of the related substances.
Background
Clotrimazole (Clotrimazole) belongs to a broad-spectrum antifungal agent of pyrrole, and is mainly used for treating deep and shallow mycoses caused by sensitive bacteria, such as cryptococcus meningitis, candida pneumonia, enteritis, histoplasmosis, tinea corporis, tinea manus and pedis, and the like. Clotrimazole has a good antibacterial effect on various fungi, especially candida albicans, and the action mechanism is to inhibit the synthesis of fungal cell membranes and influence the metabolic process of the fungi. Kemyclobutanil intermediate Z 1 (CLR-Z 1 ) Is a key intermediate in the synthesis process of clotrimazole, wherein the civilian chemical name is 1-chloro-2- (chlorodiphenylmethyl) benzene, and the molecular formula is C 19 H 14 C l2 The molecular weight is 313.22, CAS number is 42074-68-0, and the structural formula is shown in formula 1.
In the study of clotrimazole intermediate Z 1 The following impurities may be found in the process: impurity SM 1 Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d Impurity Z 1e Impurity Z 1m Impurity Z 1n Impurity Z 1i Impurity Z 1j Impurity Z 1f Impurity Z 1g . Wherein, impurity SM 1 As a key starting material, impurity Z 1a And impurity Z 1d It is a standard to produce impurities which are highly likely to occur in the process. In addition, clotrimazole intermediate Z 1 Impurity Z 1a Impurity Z 1d Impurity Z 1m Impurity Z 1i Are all easy to hydrolyze into impurity Z 1c Impurity Z 1b Impurity Z 1e Impurity Z 1n Impurity Z 1j This is the clotrimazole intermediate Z 1 The control of related impurities in the process increases a certain difficulty. Therefore, in order to ensure the quality of the clotrimazole bulk drug and the product, a clotrimazole intermediate Z is necessary 1 The above related impurities are studied and controlled.
At present, there is no literature or patent data on clotrimazole intermediate Z 1 The measurement methods of the above-mentioned related substances are reported, and therefore, it is necessary to establish a new method for realizing the clotrimazole intermediate Z 1 The related substances in the preparation are controlled so as to ensure the quality of the clotrimazole bulk drug and the preparation.
Disclosure of Invention
Accordingly, it is an object of the present invention to provide a method for preparing clotrimazole intermediate Z by high performance liquid chromatography 1 The method for separating related substances in the process has strong specificity and high sensitivity, and is a subsequent clotrimazole intermediate Z 1 The qualitative and quantitative detection of related substances in the kit is supported.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
high performance liquid chromatography is adopted for preparing clotrimazole intermediate Z 1 Method for separating related substances, which are impurities SM 1 Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d Impurity Z 1e Impurity Z 1m Impurity Z 1n Impurity Z 1i Impurity Z 1j Impurity Z 1f And impurity Z 1g Any one or more of the following; the high performance liquid chromatography is as follows: octadecylsilane chemically bonded silica is adopted as a chromatographic column filling agent, phosphoric acid aqueous solution is adopted as a mobile phase A, methanol is adopted as a mobile phase B, and a clotrimazole intermediate Z is eluted through a linear gradient 1 Wherein said related substances are separated;
the clotrimazole intermediate Z 1 The structural formula of (2) is shown as formula 1; the impurity SM 1 The structural formula of the catalyst is shown as formula 2, and the impurity Z 1a The structural formula of the catalyst is shown as formula 3, and the impurity Z 1b The structural formula of the compound is shown as formula 4Mass Z 1c The structural formula of the catalyst is shown as formula 5, and the impurity Z 1d The structural formula of the catalyst is shown as formula 6, and the impurity Z 1e The structural formula of the catalyst is shown as formula 7, and the impurity Z 1m The structural formula of the catalyst is shown as formula 8, and the impurity Z 1n The structural formula of the catalyst is shown as formula 9, and the impurity Z 1i The structural formula of the catalyst is shown as formula 10, and the impurity Z 1j The structural formula of the impurity Z is shown as formula 11 1f The structural formula of the impurity Z is shown as formula 12 1g The structural formula of (2) is shown as formula 13;
further, the program run time was 48 minutes.
Further, the sample elution sequence was: impurity Z 1a And/or impurity Z 1b Impurity Z 1d And/or impurity Z 1e Kemycin intermediate Z 1 And/or impurity Z 1c Impurity Z 1m And/or impurity Z 1n SM impurity 1 Impurity Z 1i And/or impurity Z 1j Impurity Z 1f Impurity Z 1g . The components can be characterized according to elution sequence.
Further, the gradient elution set-up procedure is as follows:
and setting the volume ratio of the mobile phase A to the mobile phase B to be 35-45:65-55;
3 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 35-45:65-55;
setting the volume ratio of the mobile phase A to the mobile phase B to be 5-15 after 35 minutes: 95-85;
setting the volume ratio of the mobile phase A to the mobile phase B to be 5-15 after 37 minutes: 95-85;
setting the volume ratio of the mobile phase A to the mobile phase B to be 35-45 after 38 minutes: 65-55;
48 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 35-45:65-55.
Preferably, the procedure for setting the gradient elution is as follows:
setting the volume ratio of the mobile phase A to the mobile phase B to be 38-42:62-58;
3 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 38-42:62-58;
35 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 10:90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 10 after 37 minutes: 90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 38-42:62-58;
48 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 38-42:62-58.
Most preferably, the gradient elution is set up as follows:
and setting the volume ratio of the mobile phase A to the mobile phase B to be 40 after 0 minutes: 60;
3 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60;
35 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 10:90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 10 after 37 minutes: 90;
38 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60;
48 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60.
further, the concentration of the mobile phase A is 0.07% -0.13%.
Preferably, the concentration of the mobile phase a is 0.09% to 0.11%, more preferably 0.1%.
Further, the flow rate of the mobile phase is 0.7-1.3ml/min; the temperature of the chromatographic column box is 17-23 ℃.
Preferably, the flow rate of the mobile phase is 0.9-1.1ml/min, more preferably 1.0ml/min; the temperature of the chromatographic column box is 18-22 ℃, more preferably 20 ℃.
Further, the sample volume was 10. Mu.l.
The second object of the invention is to provide a method for identifying the intermediate Z of clotrimazole 1 Method for determining whether related substances are contained in the composition, which can realize a clotrimazole intermediate Z 1 Effective identification of related substances in the sample.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
identification of clotrimazole intermediate Z 1 Method for preparing clotrimazole intermediate Z by using the separation method 1 Separating related substances in the water; detecting in a detector to obtain a chromatogram; the detection wavelength of the detector is 220-230nm.
Further, the retention time of each component is sequentially from short to long: impurity Z 1a And/or impurity Z 1b Impurity Z 1d And/or impurity Z 1e Kemycin intermediate Z 1 And/or impurity Z 1c Impurity Z 1m And/or impurity Z 1n SM impurity 1 Impurity Z 1i And/or impurity Z 1j Impurity Z 1f Impurity Z 1g 。
The components may be characterized in terms of retention time.
Further, the column size was 4.6mm X250 mm,5 μm; mobile phase A is phosphoric acid aqueous solution with concentration of 0.1%, mobile phase B is methanol; the flow rate is 1.0ml/min; the column degree is 20 ℃; the detection wavelength is 220-230nm;
performing linear gradient elution according to the following gradient elution program, and obtaining a chromatogram;
and setting the volume ratio of the mobile phase A to the mobile phase B to be 40 after 0 minutes: 60;
3 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60;
35 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 10:90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 10 after 37 minutes: 90;
38 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60;
48 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60.
further, the retention time was 13.6.+ -. 0.5min, which was determined as impurity Z 1a And/or impurity Z 1b The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 18.5.+ -. 0.5min, which was determined as impurity Z 1d And/or impurity Z 1e The method comprises the steps of carrying out a first treatment on the surface of the The retention time is 21.3+/-0.5 min, and the intermediate Z is judged as the clotrimazole intermediate Z 1 And/or impurity Z 1c The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 22.7.+ -. 0.5min, which was determined as impurity Z 1m And/or impurity Z 1n The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 23.4.+ -. 0.5min, which was determined as impurity SM 1 The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 24.5.+ -. 0.5min, which was determined as impurity Z 1i And/or impurity Z 1j The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 25.8.+ -. 0.5min, which was determined as impurity Z 1f The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 31.7.+ -. 0.5min, which was determined as impurity Z 1g 。
The invention further aims to provide a method for detecting the clotrimazole intermediate Z 1 Method for achieving a clotrimazole intermediate Z in 48 minutes or more than 48 minutes 1 The content detection of various related substances provides a new idea for further improving the quality of the clotrimazole bulk drug or the preparation.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
detection of clotrimazole intermediate Z 1 A method for the method content of a related substance comprising the steps of:
(1) The method is adopted to prepare the clotrimazole intermediate Z 1 Separating and detecting related substances in the sample to obtain a chromatogram;
(2) Calculating the impurity Z according to the chromatogram obtained in the step (1) by adopting an external standard method and using the peak area 1a And/or impurity Z 1b Impurity Z 1d And/or impurity Z 1e SM impurity 1 Is contained in the composition; adopts main components to self-prepareBody contrast method for calculating impurity Z 1m And/or impurity Z 1n Impurity Z 1i And/or impurity Z 1j Impurity Z 1f Impurity Z 1g Is contained in the composition.
Further, the impurity calculation formula is as follows:
1) Calculating related substances by an external standard method:
wherein A is r : the area of the impurity peak in the reference solution;
M r : weight of the reference substance;
N r : dilution of the reference substance;
p: the content of the reference substance;
A s : the area of the impurity peak in the sample solution;
M s : the weight of the test sample;
N s : dilution factor of test sample.
2) Adding correction factors to calculate related substances by a self-comparison method:
wherein A is z : the peak area of the principal component in the control solution;
A s : the area of the impurity peak in the sample solution;
f: impurity correction factor (no correction factor, in f=1);
n: dilution factors of the control solution were formulated.
Further, the sample preparation solvent is acetonitrile aqueous solution, and in the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 70:30.
preferably, the concentration of the test solution is 1mg/ml.
As a preferable technical scheme:
step 1, precisely weighing the sample, such as 25mg, placing the sample in a 25ml measuring flask, adding a solvent, performing ultrasonic dissolution, diluting to a scale, and shaking uniformly to obtain a sample solution;
step 2, precisely measuring the solution of the sample prepared in the step 1, such as 1.0ml, placing the solution in a 50ml measuring flask, diluting the solution to a scale with a solvent, and shaking the solution uniformly; precisely measuring 1.0ml, placing in a 10ml measuring flask, diluting to scale with solvent, shaking to obtain control solution;
step 3, taking the impurity SM 1 Impurity Z 1b (impurity Z) 1b Conversion to impurity Z 1a Multiplying by a factor 1.2534) with impurity Z 1e (impurity Z) 1e Conversion to impurity Z 1d Multiplying by a factor 1.0709), precisely weighing, dissolving in a solvent, and diluting to give a solution containing about the impurity SM per 1ml 1 1 μg, impurity Z 1a 2 mug and impurity Z 1d 1. Mu.g of the mixed solution as a reference solution;
step 4, precisely measuring a sample solution, a reference substance solution and a reference solution, and respectively injecting the sample solution, the reference substance solution and the reference solution into a high performance liquid chromatograph for detection to obtain a chromatogram; calculating impurity Z by using an external standard method according to the measured chromatogram 1a And/or impurity Z 1b Impurity Z 1d And/or impurity Z 1e SM impurity 1 Is contained in the composition; calculating impurity Z by adopting a main component self-comparison method 1m And/or impurity Z 1n Impurity Z 1i And/or impurity Z 1j Impurity Z 1f Impurity Z 1g Is contained in the composition.
The method hydrolyzes impurities and then feeds the samples to establish a corresponding HPLC method. Impurity Z 1a With impurity Z 1b Merging control, impurity Z 1d With impurity Z 1e Combining control, selecting impurity Z with large molecular weight and lower response value 1a And Z 1d The method is used for quantitative detection. Due to impurity Z 1a And impurity Z 1d Is easy to hydrolyze and unstable, and Z is used 1b Conversion to Z 1a Multiplying the conversion coefficient 1.2534 to prepare a reference substance solution Z 1e Conversion ofIs Z 1d The control solution is prepared by multiplying the conversion coefficient 1.0709, and the quality level is strictly controlled by calculating by an external standard method. Impurity Z 1m +Z 1n Impurity Z 1i +Z 1j Impurity Z 1f Impurity Z 1g As other single impurity controls. Impurity Z 1c Then another method study was constructed.
The invention has the beneficial effects that:
the high performance liquid chromatography provided by the invention can be used for preparing the clotrimazole intermediate Z 1 The separation and detection of various related substances possibly existing in the sample, including the impurity SM 1 Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d Impurity Z 1e Impurity Z 1m Impurity Z 1n Impurity Z 1i Impurity Z 1j Impurity Z 1f And/or impurity Z 1g . The method has the advantages of simplicity in operation, strong specificity, high sensitivity, good durability, high accuracy and the like. The method is that the clotrimazole intermediate Z 1 Provides a new method for the qualitative and quantitative determination of related substances and provides a new thought for the quality control of clotrimazole.
Drawings
FIG. 1 is a chromatogram of a mixed solution;
FIG. 2 is a chromatogram of a control solution;
FIG. 3 is a chromatogram of a test solution;
FIG. 4 is a chromatogram of a control solution;
FIG. 5 is a chromatogram of a quantitative limiting solution;
FIG. 6 is a chromatogram of a detection limit solution.
Detailed Description
The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.
In the embodiment of the invention, the structural formula of the impurity is shown in table 1.
TABLE 1 impurity Structure
EXAMPLE 1 determination of clotrimazole intermediate Z by high Performance liquid chromatography 1 Related substances in (a)
1. Preparing a sample to be tested
Solvent: acetonitrile aqueous solution, by acetonitrile and water according to the volume ratio 70:30.
Test solution: taking 25mg of the product, precisely weighing, placing into a 25ml measuring flask, adding solvent, performing ultrasonic dissolution, diluting to a scale, and shaking uniformly to obtain the product.
Control solution: precisely measuring 1.0ml of the sample solution, placing in a 50ml measuring flask, diluting to scale with solvent, and shaking; precisely weighing 1.0ml, placing in a 10ml measuring flask, diluting to scale with solvent, and shaking.
Control solution: taking impurity CLR-SM 1 Impurity CLR-Z 1b (impurity CLR-Z) 1b Conversion to impurity CLR-Z 1a Multiplying by a coefficient 1.2534) with impurity CLR-Z 1e (impurity CLR-Z) 1e Conversion to impurity CLR-Z 1d Multiplying by a factor 1.0709), precisely weighing, dissolving in a solvent, and diluting to obtain a solution containing about impurity CLR-SM per 1ml 1 1 μg, impurity CLR-Z 1a 2 μg and impurity CLR-Z 1d 1 μg of the mixed solution.
2. Test conditions: as shown in tables 2 and 3.
TABLE 2 test conditions
TABLE 3 gradient elution program table
Time (minutes) Mobile phase a (%) Mobile phase B (%)
0 40 60
3 40 60
35 10 90
37 10 90
38 40 60
48 40 60
3. Measurement method
Precisely measuring the sample solution, the reference substance solution and the reference solution, respectively injecting into a liquid chromatograph, detecting according to the chromatographic conditions in the step 2, and recording the chromatograms. Impurity CLR-Z in the chromatogram of the reference solution 1a Impurity CLR-Z 1d Impurity CLR-SM 1 And (5) sequentially generating peaks.
Limit: respectively calculating impurity CLR-Z according to an external standard method except peak area 1a Impurity CLR-Z 1d Impurity CLR-SM 1 Content, main component self-comparison method calculates other single impurity content, impurity CLR-Z 1m Impurity CLR-Z 1n Impurity CLR-Z 1i Impurity CLR-Z 1j Impurity CLR-Z 1f Impurity CLR-Z 1g As other single impurity controls. The impurity limits are set forth in Table 4 below with the impurity peaks less than 0.05% being ignored.
TABLE 4 Table 4
Impurity name Limit of impurity
Impurity CLR-SM 1 0.10％
Impurity CLR-Z 1a 0.2％
Impurity CLR-Z 1d 0.10％
Other single impurities 0.5％
Note that: impurity CLR-Z 1a Control of impurity CLR-Z for incorporation 1a (molecular weight 271.57) and impurity CLR-Z 1b (molecular weight 216.66); impurity CLR-Z 1d Control of impurity CLR-Z for incorporation 1d (molecular weight 278.78) and impurity CLR-Z 1e (molecular weight 260.33).
EXAMPLE 2 specificity
1. Preparing a solution:
solvent: acetonitrile aqueous solution, by acetonitrile and water according to the volume ratio 70:30.
Impurity CLR-SM1 stock solution: precisely weighing impurity CLR-SM 1 25mg, placing in a 25ml measuring flask, adding solvent, diluting to scale, and shaking.
Impurity CLR-Z 1a Stock solution: precisely weighing impurity CLR-Z 1a 25mg, placing in a 25ml measuring flask, adding solvent, diluting to scale, and shaking.
Impurity CLR-Z 1d Stock solution: precisely weighing impurity CLR-Z 1d 25mg, putting into a 25ml measuring flask, adding solvent, dissolving by ultrasonic, diluting to scale, and shaking uniformly to obtain the final product.
Impurity CLR-Z 1b Stock solution: precisely weighing impurity CLR-Z 1b 25mg, placing in a 25ml measuring flask, adding solvent, diluting to scale, and shaking.
Impurity CLR-Z 1e Stock solution: precisely weighing impurity CLR-Z 1e 25mg, putting into a 25ml measuring flask, adding solvent, dissolving by ultrasonic, diluting to scale, and shaking uniformly to obtain the final product.
Impurity CLR-Z 1c Stock solution: precisely weighing impurity CLR-Z 1c 25mg, putting into a 25ml measuring flask, adding solvent, dissolving by ultrasonic, diluting to scale, and shaking uniformly to obtain the final product.
Impurity CLR-Z 1i Stock solution: precisely weighing impurity CLR-Z 1i 25mg, placing in a 25ml measuring flask, adding solvent, diluting to scale, and shaking.
Impurity CLR-Z 1j Stock solution: precisely weighing impurity CLR-Z 1j 25mg, put into a 25ml measuring flask, and diluted with solvent to the scaleShaking to obtain the final product.
Impurity CLR-Z 1m Stock solution: precisely weighing impurity CLR-Z 1m 25mg, placing in a 25ml measuring flask, adding solvent, dissolving with ultrasound, diluting to scale, and shaking.
Impurity CLR-Z 1n Stock solution: precisely weighing impurity CLR-Z 1n 25mg, placing in a 25ml measuring flask, adding solvent, diluting to scale, and shaking.
Impurity CLR-Z 1f Stock solution: precisely weighing impurity CLR-Z 1f 25mg, placing in a 25ml measuring flask, adding solvent, diluting to scale, and shaking.
Impurity CLR-Z 1g Stock solution: precisely weighing impurity CLR-Z 1g 25mg, putting into a 25ml measuring flask, adding solvent, dissolving by ultrasonic, diluting to scale, and shaking uniformly to obtain the final product.
Impurity control stock solution: precise impurity CLR-SM measuring 1 Stock solution, impurity CLR-Z 1d 1ml of each stock solution, impurity CLR-Z 1a 2ml of stock solution is put into a same 100ml measuring flask, diluted to the scale by solvent and shaken uniformly to obtain the product.
Other impurity mixing stock: precise impurity CLR-Z measuring 1c Stock solution, impurity CLR-Z 1i Stock solution, impurity CLR-Z 1j Stock solution, impurity CLR-Z 1m Stock solution, impurity CLR-Z 1n Stock solution, impurity CLR-Z 1f Stock solution, impurity CLR-Z 1g And (5) storing the stock solution, placing the stock solution into a same 100ml measuring flask, diluting the stock solution to a scale by using a solvent, and shaking the stock solution uniformly to obtain the finished product.
Mixing solution: precisely weighing sample Z 1 25mg, placing in a 25ml measuring flask, precisely measuring 2.5ml of impurity reference stock solution and 2.5ml of other impurity mixed stock solution, placing in the same 25ml measuring flask, adding solvent, ultrasonically dissolving, diluting to scale, and shaking.
2. Detection of
And (3) respectively injecting the solutions prepared in the step (1) into a high performance liquid chromatograph for detection, and obtaining a chromatogram.
Results: as shown in tables 5 and 6 and FIGS. 1 to 4, the blank solution, the sample solution and other impurities do not interfere with the impurity CLR-SM 1 Impurity CLR-Z 1a Impurity CLR-Z 1d Is measured. The minimum separation degree between each target impurity peak and the adjacent peak, and between the main peak and the adjacent impurity peak is more than 1.5.
TABLE 5 determination of the specificity test
TABLE 6 response value comparison
Example 3 System applicability
1. Solution preparation
Impurity control stock solution: the "impurity control stock solution" under the "example 2. Proprietary" item was prepared.
Sample stock solution i: precisely weighing 500mg of the sample, placing in a 50ml measuring flask, adding solvent, dissolving with ultrasound, diluting to scale, and shaking.
Sample stock solution ii: precisely measuring 1ml of sample stock solution I, placing into a 10ml measuring flask, adding solvent to dilute to scale, and shaking to obtain the final product.
Sample stock solution iii: precisely measuring 1ml of sample stock solution II, placing into a 50ml measuring flask, diluting with solvent to scale, and shaking.
Control solution: precisely measuring 1ml of sample stock solution III, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking.
Control solution: the "control solution" under the "accuracy" term was formulated as in "example 6".
2. Detection of
Taking control solution and control solution, continuously sampling for 6 times, recording a chromatogram, and observing the main peak area RSD, the theoretical plate number and the tailing factor.
Results: as shown in tables 7 and 8. The control solution was continuously sampled 6 times, the main peak area RSD was 0.2% (n=6), the peak area RSD is less than 2.0%; the number of theoretical plates is larger than 3000, and the tailing factors of the main peaks are smaller than 2.0. The control solution was continuously sampled 6 times, impurity CLR-Z 1a Impurity CLR-Z 1d Impurity CLR-SM 1 The peak areas RSD are respectively 0.6%, 0.5% and 0.7%, the theoretical plate number is more than 3000, and the main peak tailing factors are less than 2.0.
TABLE 7 control solution System applicability results
Number of sample injections Retention time (min) Main peak area Tailing factor Number of theoretical plates
1 21.37 0.648 1.07 60158
2 21.39 0.647 1.07 59652
3 21.318 0.645 1.07 60180
4 21.315 0.646 1.07 59408
5 21.4 0.645 1.07 60601
6 21.303 0.646 1.08 58734
Average n=6 21.349 0.646 —— ——
RSD 0.3 0.2 —— ——
TABLE 8 control solution System applicability results
Example 4 quantitative limit and detection limit
1. Preparing a solution:
impurity stock solution: same as in example 2 proprietary under item impurity CLR-SM 1 Stock solution "," impurity CLR-Z 1a Stock solution "," impurity CLR-Z 1d The stock solution is prepared.
Sample stock solution: the same "example 3. System applicability" under the term "test sample stock solution II" was prepared.
Quantitatively limiting stock solution: precisely measuring sample stock solution and impurity CLR-SM 1 Stock solution, impurity CLR-Z 1a Stock solution, impurity CLR-Z 1d And (3) respectively placing 1ml of the stock solution into a 50ml measuring flask, diluting to a scale with a solvent, and shaking uniformly to obtain the product. (20. Mu.g/ml)
Quantitative limiting solution: precisely measuring 1ml of quantitative limited stock solution, placing into a 50ml measuring flask, diluting to scale with solvent, and shaking. (0.4. Mu.g/ml)
Detection limit solution: precisely measuring 3ml of quantitative limiting solution, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking.
(0.12μg/ml)
2. Measurement method
Taking the quantitative limiting solution to continuously sample for 6 times, detecting the limiting solution to continuously sample for 3 times, calculating the ratio (signal to noise ratio) of the peak height to the noise of each peak, and recording a chromatogram.
Results: as shown in tables 9 to 10 and fig. 5 to 6. Impurity CLR-Z 1a The quantitative limit concentration of (2) was 0.3969. Mu.g/ml, expressed as concentration present in the sample as 0.04%, the peak area RSD as 2.5% and the signal to noise ratio average as 13.3; impurity CLR-Z 1d The quantitative limit concentration of (2) was 0.4118. Mu.g/ml, which was 0.04% as the concentration present in the sample, 1.9% as the peak area RSD, and 14.5 as the signal-to-noise ratio average; CLR-Z 1 The quantitative limit concentration of (2) was 0.4005. Mu.g/ml, and the concentration was 0.04% as the concentration present in the sample, peakThe area RSD is 1.0%, and the signal to noise ratio average value is 21.1; impurity CLR-SM 1 The quantitative limit concentration of (2) is 0.3928 μg/ml, expressed as concentration present in the sample is 0.04%, the peak area RSD is 1.5%, and the signal to noise ratio average is 20.0; indicating that the impurity content in the sample was quantitatively detected at 0.04%.
Impurity CLR-Z 1a The detection limit concentration of (2) is 0.1191 mug/ml, the concentration in the sample is 0.01%, and the signal to noise ratio average value is 3.7; impurity CLR-Z 1d The detection limit concentration of (2) is 0.1235 mug/ml, the concentration in the sample is 0.01%, and the signal-to-noise ratio average value is 4.0; impurity CLR-Z 1 The detection limit concentration of (2) is 0.1201 mug/ml, the concentration in the sample is 0.01%, and the signal to noise ratio average value is 6.2; impurity CLR-SM 1 The detection limit concentration of (2) was 0.1178. Mu.g/ml, which was 0.01% as the concentration present in the sample, and the signal-to-noise ratio was 5.4 as the average. Indicating that the impurity content in the sample was 0.01%.
TABLE 9 quantitative limit determination results
TABLE 10 detection limit measurement results
EXAMPLE 5 precision
1. Preparation and detection of precision solution
Control solution: the same "example 6. Accuracy" under item "control solution" was prepared.
Impurity control stock solution: the same example 6. Accuracy "under term" impurity control stock solution "was prepared.
Adding a labeled test sample solution: taking CLR-Z 1 About 25mg, precisely weighed,placing into 25ml measuring flask, precisely measuring 2.5ml of impurity reference substance stock solution, placing into the same 25ml measuring flask, adding solvent, ultrasonic dissolving, diluting to scale, and shaking. (parallel preparation of 6 parts)
Control solution: precisely measuring 1ml of the solution of the sample to be marked, placing the solution into a 50ml measuring flask, diluting the solution to a scale with a solvent, and shaking the solution evenly; precisely measuring 1ml, placing in a 10ml measuring flask, diluting with solvent to scale, and shaking.
Taking control solution, continuously injecting 6 needles, adding the standard sample solution and the control solution, measuring according to the chromatographic conditions, and recording the chromatogram. Respectively calculating impurity CLR-Z according to an external standard method except peak area 1a Impurity CLR-Z 1d Impurity CLR-SM 1 The content of unknown impurities is calculated by the main component self-comparison method, and the content of related substances and RSD in 6 parts of sample solution are calculated.
2. Intermediate precision solution preparation
To examine the effect of random variation factors on precision, different analysts test at different times and with different instruments.
Z 1b And Z 1e Stock solution: precisely weighing impurity CLR-Z 1b 40mg, impurity CLR-Z 1e 25mg, putting into a same 25ml measuring flask, adding solvent, dissolving by ultrasonic, diluting to scale, and shaking uniformly to obtain the final product.
SM 1 Stock solution: precisely weighing impurity CLR-SM 1 25mg, placing in a 25ml measuring flask, adding solvent, diluting to scale, and shaking.
Z 1a And Z 1d Stock solution: precisely weighing impurity CLR-Z 1a 50mg, impurity CLR-Z 1d 25mg, putting into a same 25ml measuring flask, adding solvent, dissolving by ultrasonic, diluting to scale, and shaking uniformly to obtain the final product.
Control solution: precise measuring Z 1b And Z 1e Stock solution 1ml, SM 1 1ml of stock solution is put into a same 100ml measuring flask, diluted to a scale by adding solvent and uniformly shaken; precisely measuring 1ml, placing in a 10ml measuring flask, diluting with solvent to scale, and shaking.
Impurity control stock solution: precise measuring Z 1a And Z 1d Stock solution 1ml, SM 1 And (3) placing 1ml of stock solution into a same 100ml measuring flask, adding a solvent to dilute to a scale, and shaking uniformly to obtain the product.
Adding a labeled test sample solution: taking CLR-Z 1 About 25mg, precisely weighing, placing into 25ml measuring flask, precisely measuring 2.5ml of impurity reference substance stock solution, placing into the same 25ml measuring flask, adding solvent, ultrasonically dissolving, diluting to scale, and shaking. (parallel preparation of 6 parts)
Control solution: precisely measuring 1ml of the solution of the sample to be marked, placing the solution into a 50ml measuring flask, diluting the solution to a scale with a solvent, and shaking the solution evenly; precisely measuring 1ml, placing in a 10ml measuring flask, diluting with solvent to scale, and shaking.
Taking control solution, continuously sampling for 6 times, taking the sample solution and the control solution, measuring according to the chromatographic conditions, and recording the chromatogram. Respectively calculating impurity CLR-Z according to an external standard method except peak area 1a Impurity CLR-Z 1d Impurity CLR-SM 1 The content of unknown impurities is calculated by the main component self-comparison method, and the content of related substances and RSD in 6 parts of sample solution are calculated. And calculating RSD of the related substance content of the 12 samples with precision.
Results: as shown in Table 11, the impurity CLR-Z in the sample solution was added with 12 parts of the standard sample solution with precision and intermediate precision 1a The average content is 0.20%, and RSD is 1.5%; impurity CLR-Z 1d The average content is 0.10% and the RSD is 3.9%; impurity CLR-SM 1 The average content is 0.10% and the RSD is 3.0%; the average value of the other individual impurity (RRT 1.47) content was 0.05%. The average content of the detected total impurities was 0.51%, and the RSD was 1.8%. The precision test and the intermediate precision test of the review method and the results thereof meet the requirements, and the detection method is suitable for the inspection of related substances of the product.
TABLE 11 precision test measurement results
Example 6 accuracy
1. Solution preparation
Z 1b And Z 1e Stock solution: precisely weighing impurity CLR-Z 1b 40mg, impurity CLR-Z 1e 25mg, putting into a same 25ml measuring flask, adding solvent, dissolving by ultrasonic, diluting to scale, and shaking uniformly to obtain the final product.
SM 1 Stock solution: precisely weighing impurity CLR-SM 1 25mg, placing in a 25ml measuring flask, adding solvent, diluting to scale, and shaking.
Z 1a And Z 1d Stock solution: precisely weighing impurity CLR-Z 1a 50mg, impurity CLR-Z 1d 25mg, putting into a same 25ml measuring flask, adding solvent, dissolving by ultrasonic, diluting to scale, and shaking uniformly to obtain the final product.
Impurity control stock solution: precise measuring Z 1a And Z 1d Stock solution 1ml, SM 1 And (3) placing 1ml of stock solution into a same 100ml measuring flask, adding a solvent to dilute to a scale, and shaking uniformly to obtain the product.
Sample stock solution: taking 250mg of a test sample, precisely weighing, placing into a 25ml measuring flask, adding a solvent, performing ultrasonic dissolution, diluting to a scale, and shaking uniformly to obtain the product.
Unlabeled solution: precisely measuring 1ml of sample stock solution, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking.
Control solution: precise measuring Z 1b And Z 1e Stock solution 1ml, SM 1 1ml of stock solution is put into a same 100ml measuring flask, diluted to a scale by adding solvent and uniformly shaken; precisely measuring 1ml, placing in a 10ml measuring flask, diluting with solvent to scale, and shaking.
80% of the labeling solution: precisely measuring 1.0ml of sample stock solution, 0.8ml of impurity reference sample stock solution, placing into the same 10ml measuring flask, diluting with solvent to scale, and shaking. (parallel preparation of 3 parts)
100% of the labeling solution: precisely measuring 1.0ml of sample stock solution, 1.0ml of impurity reference sample stock solution, placing into the same 10ml measuring flask, diluting with solvent to scale, and shaking. (parallel preparation of 3 parts)
120% of labeling solution: precisely measuring 1.0ml of sample stock solution, 1.2ml of impurity reference sample stock solution, placing into the same 10ml measuring flask, diluting with solvent to scale, and shaking. (parallel preparation of 3 parts)
2. Detection of
Respectively taking 80% labeled solution, 100% labeled solution, 120% labeled solution, unlabeled solution and reference solution, precisely measuring 10 μl according to the above chromatographic conditions, and injecting into a liquid chromatograph for recording chromatogram. And calculating the recovery rate of each impurity according to an external standard method.
Results: as shown in Table 12, impurity CLR-Z 1a The average recovery rate is 102.1% and RSD is 0.6%; impurity CLR-Z 1d The average recovery rate is 103.2%, and the RSD is 0.9%; impurity CLR-SM 1 The average recovery rate was 100.1% and RSD was 2.4%. The recovery rate is between 90% and 108%, which shows that the measurement of the accuracy of each impurity meets the requirements.
TABLE 12 accuracy test measurement results
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Example 7 durability
Mixing solution: the same "example 1. Proprietary" under item "mixed solution" was formulated.
The mixed solution is taken and tested by using normal mobile phase chromatographic conditions and adjusting chromatographic conditions (column temperature + -2 ℃, flow rate + -0.1 ml/min, mobile phase phosphoric acid content + -0.01% and mobile phase initial proportion + -2%), after the instrument system is stable, the mixed solution is tested respectively, and the separation degree between peaks and the relative retention time of known impurities are recorded.
Results: as shown in tables 13-15, when the flow rate, column temperature, mobile phase proportion and mobile phase phosphoric acid content slightly fluctuate, the minimum separation degree between each impurity peak and the adjacent peak is more than 1.5, and the method has good durability and meets the requirements. The relative retention time of each known impurity is within +/-0.03, and the method has good durability.
TABLE 13 chromatographic System durability determination results-minimum separation from adjacent peaks
TABLE 14 chromatographic System durability determination results-retention time
TABLE 15 chromatographic System durability determination results-relative retention time
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Claims (10)

1. High performance liquid chromatography is adopted for preparing clotrimazole intermediate Z 1 A method for separating related substances, characterized in that the related substances are impurities SM 1 Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d Impurity Z 1e Impurity Z 1m Impurity Z 1n Impurity Z 1i Impurity Z 1j Impurity Z 1f And impurity Z 1g Any one or more of the following; the high performance liquid chromatography is as follows: octadecylsilane chemically bonded silica is adopted as a chromatographic column filling agent, phosphoric acid aqueous solution is adopted as a mobile phase A, methanol is adopted as a mobile phase B, and a clotrimazole intermediate Z is eluted through a linear gradient 1 Wherein said related substances are separated;
the clotrimazole intermediate Z 1 The structural formula of (2) is shown as formula 1; the impurity isMass SM 1 The structural formula of the catalyst is shown as formula 2, and the impurity Z 1a The structural formula of the catalyst is shown as formula 3, and the impurity Z 1b The structural formula of the catalyst is shown as formula 4, and the impurity Z 1c The structural formula of the catalyst is shown as formula 5, and the impurity Z 1d The structural formula of the catalyst is shown as formula 6, and the impurity Z 1e The structural formula of the catalyst is shown as formula 7, and the impurity Z 1m The structural formula of the catalyst is shown as formula 8, and the impurity Z 1n The structural formula of the catalyst is shown as formula 9, and the impurity Z 1i The structural formula of the catalyst is shown as formula 10, and the impurity Z 1j The structural formula of the impurity Z is shown as formula 11 1f The structural formula of the impurity Z is shown as formula 12 1g The structural formula of (2) is shown as formula 13;
2. the method of claim 1, wherein the gradient elution set-up procedure is as follows:
and setting the volume ratio of the mobile phase A to the mobile phase B to be 35-45:65-55;
3 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 35-45:65-55;
setting the volume ratio of the mobile phase A to the mobile phase B to be 5-15 after 35 minutes: 95-85;
setting the volume ratio of the mobile phase A to the mobile phase B to be 5-15 after 37 minutes: 95-85;
setting the volume ratio of the mobile phase A to the mobile phase B to be 35-45 after 38 minutes: 65-55;
48 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 35-45:65-55.
3. The method according to claim 1, wherein the concentration of mobile phase a is 0.07% to 0.13%.
4. The method according to claim 1, wherein the flow rate of the mobile phase is 0.7-1.3ml/min; the temperature of the chromatographic column box is 17-23 ℃.
5. Identification of clotrimazole intermediate Z 1 A process for the preparation of clotrimazole intermediates Z, characterized in that a process according to any of claims 1 to 4 is used 1 Separating related substances in the water; detecting in a detector to obtain a chromatogram; the detection wavelength of the detector is 220-230nm.
6. The method of claim 5, wherein the retention time of each component is, in order from short to long: impurity Z 1a And/or impurity Z 1b Impurity Z 1d And/or impurity Z 1e Kemycin intermediate Z 1 And/or impurity Z 1c Impurity Z 1m And/or impurity Z 1n SM impurity 1 Impurity Z 1i And/or impurity Z 1j Impurity Z 1f Impurity Z 1g 。
7. The method of claim 5, wherein the chromatographic column format is 4.6mm x 250mm,5 μm; mobile phase A is phosphoric acid aqueous solution with concentration of 0.1%, mobile phase B is methanol; the flow rate is 1.0ml/min; the column degree is 20 ℃;
the detection wavelength is 220-230nm;
performing linear gradient elution according to the following gradient elution program, and obtaining a chromatogram;
and setting the volume ratio of the mobile phase A to the mobile phase B to be 40 after 0 minutes: 60;
3 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60;
35 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 10:90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 10 after 37 minutes: 90;
38 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60;
48 minutes, setting the volume ratio of the mobile phase A to the mobile phase B as 40:60.
8. the method according to claim 7, wherein the retention time is 13.6.+ -. 0.5min, determined as impurity Z 1a And/or impurity Z 1b The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 18.5.+ -. 0.5min, which was determined as impurity Z 1d And/or impurity Z 1e The method comprises the steps of carrying out a first treatment on the surface of the The retention time is 21.3+/-0.5 min, and the intermediate Z is judged as the clotrimazole intermediate Z 1 And/or impurity Z 1c The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 22.7.+ -. 0.5min, which was determined as impurity Z 1m And/or impurity Z 1n The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 23.4.+ -. 0.5min, which was determined as impurity SM 1 The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 24.5.+ -. 0.5min, which was determined as impurity Z 1i And/or impurity Z 1j The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 25.8.+ -. 0.5min, which was determined as impurity Z 1f The method comprises the steps of carrying out a first treatment on the surface of the The retention time was 31.7.+ -. 0.5min, which was determined as impurity Z 1g 。
9. Detection of clotrimazole intermediate Z 1 A method for the method content of the related substances, characterized by comprising the following steps:
(1) A clotrimazole intermediate Z by the method of any one of claims 5-8 1 Separating and detecting related substances in the sample to obtain a chromatogram;
(2) Calculating the impurity Z according to the chromatogram obtained in the step (1) by adopting an external standard method and using the peak area 1a And/or impurity Z 1b Impurity Z 1d And/or impurity Z 1e SM impurity 1 Is contained in the composition; calculating impurity Z by adopting a main component self-comparison method 1m And/or impurity Z 1n Impurity Z 1i And/or impurity Z 1j Impurity Z 1f Impurity Z 1g Is contained in the composition.
10. The method of claim 9, wherein the sample formulation solvent is an aqueous acetonitrile solution in which the volume ratio of acetonitrile to water is 70:30.
CN202311776084.3A 2023-12-21 2023-12-21 Isolation assay of clotrimazole intermediate Z 1 High performance liquid chromatography of related substances in (B) Pending CN117825547A (en)

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