CN114252536A - High performance liquid chromatography method for determining related substances in bifonazole bulk drug - Google Patents
High performance liquid chromatography method for determining related substances in bifonazole bulk drug Download PDFInfo
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Abstract
The invention provides a high performance liquid chromatography method for determining related substances in a bifonazole raw material medicine. The high performance liquid chromatography method for measuring related substances in the bifonazole raw material medicine comprises the following steps: 1) dissolving a to-be-detected sample of the bifonazole bulk drug in a mobile phase, and filtering to obtain a to-be-detected solution; 2) the method comprises the steps of adopting a mixed solution of a buffer solution and a solvent as a mobile phase, measuring a standard solution and a solution to be measured by using a high performance liquid chromatography to obtain a standard curve, and analyzing the content of bifonazole and related substances in the solution to be measured according to the standard curve. The high performance liquid chromatography method has the advantages of good separation selectivity, symmetrical peak shape, linear quantitative curve, good repeatability, and stable retention time, separation degree and signal response value.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and relates to a high performance liquid chromatography method for determining related substances in a bifonazole bulk drug.
Background
Bifonazole, chemically known as 1- (rho, alpha-diphenylbenzyl) imidazole, is an imidazole derivative, is an imidazole antifungal drug free of halogen, is odorless, slightly soluble in methanol or absolute ethanol and almost insoluble in water, and has the pharmacological actions of: the bifonazole has the effects of broad-spectrum resisting dermatophytes, yeasts, filamentous fungi and biphase fungi, has stronger antibacterial activity, is also effective to malassezia furfur and gram-positive cocci, and has good external application effect on animal experimental dermatophytes; it has broad antibacterial spectrum, has 90% of pathogenic fungi inhibiting effect, and can inhibit various dermatophytes (such as Trichophyton, Microsporum), yeasts (chemical book, such as Candida, Calycopsis, and Torulopsis glabrata), molds (such as Aspergillus), and various fungi (such as Coccidioides, Blastomyces, and Histoplasma).
The quality standards of the biphenyl benzyl azole bulk drugs are recorded in the China pharmacopoeia 2020 edition (second part), the United states pharmacopoeia 36 edition and the European pharmacopoeia 8.0 edition, and the related substances are biphenyl benzyl alcohol remained in the synthesis process. Referring to a method for testing related substances of bifonazole bulk drug in the second department of Chinese pharmacopoeia (2020 edition): the biphenyl benzyl azole is measured by a high performance liquid phase method by using methanol/water/tetrahydrofuran (84/15/1) (v/v/v) as a mobile phase.
However, the current chinese pharmacopoeia method has the specific challenges: the analytical method is unstable and is reflected in the peak shape and retention time of bifonazole: 1) the peak shapes of bifonazole are poor by the same mobile phase preparation method of different types of C18 reversed phase chromatographic columns; 2) the same chromatographic method is used for the same bottle of mobile phase, continuous sample introduction is carried out, and the reproducibility of the bifonazole is poor; 3) the same chromatographic column and the same mobile phase preparation method are adopted, but the separation reproducibility of different water sources is poor. The tracing analysis is as follows: 1) imidazole functional groups in the biphenyl benzyl azole structure can be protonated under acidic conditions, so that the biphenyl benzyl azole structure is positively charged and can generate charge interaction with silicon hydroxyl on the surface of a chromatographic packing of a silica gel matrix; 2) the mobile phase uses a pure water system, and the pH is not controlled, so that the charge state of the silicon hydroxyl and the bifonazole on the surface of the stationary phase is unstable, and the stability of the peak shape and the retention time is influenced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the high performance liquid chromatography method for measuring the related substances in the bifonazole bulk drug, which has the advantages of good separation selectivity, symmetrical peak shape, linear quantitative curve, good repeatability, stable retention time, separation degree and signal response value.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high performance liquid chromatography method for measuring related substances in a bifonazole raw material medicine comprises the following steps:
1) dissolving a to-be-detected sample of the bifonazole bulk drug in a mobile phase, and filtering to obtain a to-be-detected solution;
2) the method comprises the steps of adopting a mixed solution of a buffer solution and a solvent as a mobile phase, measuring a standard solution and a solution to be measured by using a high performance liquid chromatography to obtain a standard curve, and analyzing the content of bifonazole and related substances in the solution to be measured according to the standard curve.
The high performance liquid chromatography method for measuring related substances in the bifonazole bulk drug adopts the mixed solution of the buffer solution and the solvent as the mobile phase, and the buffer solution replaces pure water in the mobile phase, so that the peak shape, the column effect and the reproducibility of the bifonazole are obviously improved.
In step 2), the buffer solution in the mobile phase is an acetate buffer solution or a phosphate buffer solution, and preferably is an acetate buffer solution.
Preferably, the solvent is an organic solvent.
Preferably, the organic solvent is methanol.
In the step 2), the mobile phase is a mixed solution of acetate buffer solution and methanol.
In the step 2), the volume ratio of the acetate buffer solution to the methanol is (80-90) to (10-20), and preferably 85: 15.
In the step 2), the acetate in the acetate buffer solution comprises ammonium acetate, sodium acetate, potassium acetate and hydrates thereof.
In the step 2), the chromatographic column adopted by the high performance liquid chromatography is a reversed phase liquid chromatographic column, and preferably a C18 chromatographic column.
Preferably, the C18 chromatographic column is an uncapped single-point bonded side chain protected C18 chromatographic column, preferably a chromocore AR C18 chromatographic column. Wherein, the Chromcore AR C18 is formed by connecting a side chain protection C18 silanization reagent to monodisperse high-purity porous silica gel through a covalent bond, has good acid resistance and water resistance, and stronger surface polarity, and shows unique separation selectivity.
In step 1), the pH of the mobile phase is 5-5.4, for example, pH5, 5.1, 5.2, 5.3, 5.4, etc.; preferably 5.2.
In the step 2), the detection conditions of the high performance liquid chromatography are as follows: column temperature of the column: 20-30 deg.C, such as 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C, preferably 25 deg.C; the flow rate of the mobile phase is 0.9-1.1mL/min, and the detection wavelength is 252-256 nm.
In the step 1), the solvent is a mixed solution of methanol and water with a volume ratio of (80-90) to (10-20), preferably a mixed solution of methanol and water with a volume ratio of 85: 15.
Related substances in the bifonazole bulk drug comprise biphenyl benzyl alcohol.
Compared with the prior art, the invention has the beneficial effects that:
the high performance liquid chromatography method for measuring related substances in the bifonazole bulk drug has the advantages of good separation selectivity, symmetrical peak shape, linear quantitative curve, good repeatability and stable retention time, separation degree and signal response value. Specifically, under the preferable chromatographic conditions, the sample injection (10 needles) is repeated for biphenyl benzyl alcohol (5 μ g/mL) and biphenyl benzyl azole (10 μ g/mL) control solutions, the retention time RSD of the biphenyl benzyl alcohol is 0.027%, the peak area RSD is 0.129%, the retention time RSD of the biphenyl benzyl azole is 0.028%, the peak area RSD is 0.038%, and the reproducibility is good; the separation degree of the biphenyl benzyl alcohol and the biphenyl benzyl azole is 7.70, the biphenyl benzyl alcohol is in the concentration range of 1.00-10.0 mu g/mL, the linear equation is that y is 1.1639x-0.014, the correlation coefficient R2 is 0.9999, the linear relation between the peak area and the concentration is good, the detection limit is 3.3ng/mL, and the quantification limit is 11.1 ng/mL; the concentration of the bifonazole is within the range of 2.00-20.0 mug/mL, the linear equation is that y is 0.7789x-0.0898, the correlation coefficient R2 is 0.9996, the linear relation between the peak area and the concentration is good, the detection limit is 15.3ng/mL, and the quantification limit is 51.0 ng/mL.
Drawings
FIG. 1 is a high performance liquid chromatogram of example 1 of the present invention;
FIG. 2 is a high performance liquid chromatogram of example 2 of the present invention;
FIG. 3 is a high performance liquid chromatogram of example 3 of the present invention;
FIG. 4 is a high performance liquid chromatogram of example 4 of the present invention;
FIG. 5 is a high performance liquid chromatogram of example 5 of the present invention;
FIG. 6 is a high performance liquid chromatogram of example 6 of the present invention;
FIG. 7 is a high performance liquid chromatogram of example 7 of the present invention;
FIG. 8 is a graphical representation of the regression equation and linear range for biphenyl benzyl alcohol of example 9 of the present invention;
FIG. 9 is a schematic diagram of the regression equation and linear range of bifonazole of example 9 of the present invention;
FIG. 10 is a high performance liquid chromatogram of example 10 of the present invention;
FIG. 11 is a high performance liquid chromatogram of comparative example 1 of the present invention;
FIG. 12 is a high performance liquid chromatogram of comparative example 2 of the present invention;
FIG. 13 is a high performance liquid chromatogram of comparative example 3 of the present invention;
FIG. 14 is a high performance liquid chromatogram of comparative example 4 of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments.
Unless otherwise specified, various starting materials of the present invention are commercially available or prepared according to conventional methods in the art.
The high performance liquid chromatography method for measuring related substances in the bifonazole raw material medicine comprises the following steps:
1) dissolving a to-be-detected sample of the bifonazole bulk drug in a mobile phase, and filtering to obtain a to-be-detected solution;
2) the method comprises the steps of adopting a mixed solution of a buffer solution and a solvent as a mobile phase, measuring a standard solution and a solution to be measured by using a high performance liquid chromatography to obtain a standard curve, and analyzing the content of bifonazole and related substances in the solution to be measured according to the standard curve.
The invention also comprises the preparation of the following solutions:
1. system applicability solution: proper amounts of the bifonazole and the bifonazole are taken, and the solution containing 1mg of the bifonazole and 5 mu g of the bifonazole in each 1mL is prepared by adding the mobile phase for dissolving and diluting.
2. Test solution: taking a proper amount of a sample to be detected, precisely weighing, adding a mobile phase for dissolving, and quantitatively diluting to prepare a solution containing about 1mg of bifonazole in every 1 mL.
3. Control solution: precisely measuring 1mL of the test solution, placing the test solution in a 100mL measuring flask, diluting the test solution to a scale with a mobile phase, and shaking up.
4. Sensitivity solution: the test solution was diluted 2000-fold with the mobile phase.
The mobile phase was a mixture of 85/15 vol% methanol and 20mM ammonium acetate, and the pH of the mobile phase was 5.2.
In the following examples, the apparatus used was a high performance liquid chromatography Thermo Fisher Scientific UltiMate 3000 liquid chromatograph.
The invention adopts two buffer systems, namely a phosphate buffer system and an acetate buffer system, and specifically comprises the following steps:
the phosphate buffer system is 10mM ammonium dihydrogen phosphate, pH3.0, and is prepared by the following method: 1000. + -. 1.0g of deionized water was weighed, 1.15g of ammonium dihydrogen phosphate was added, and after dissolution, the pH was adjusted to 3.0 with phosphoric acid.
The acetate buffer system is 20mM ammonium acetate, pH5.2, and is prepared as follows: 1000. + -. 1.0g of deionized water was weighed, 1.54g of ammonium acetate was added, dissolved and adjusted to pH5.2 with glacial acetic acid.
Example 1
Based on the "chinese pharmacopoeia" 2020 edition (second part), the present example uses a phosphate buffer system, the same mobile phase, and different types of reverse phase C18 chromatographic columns for testing, and the high performance liquid chromatogram is shown in fig. 1, and the test results are shown in table 1.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: single point bonding with end capping C18 (4.6X 250mm, 5 μm);
non-end-capped C18 with single point linkage of side chain protection (4.6X 250mm, 5 μm);
multi-point bonding with end capping C18 (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/10 mM ammonium dihydrogen phosphate, pH 3.0;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: control solution.
TABLE 1
As can be seen from Table 1, the peak shape of bifonazole (peak 1) is symmetrical and the column efficiency is high when three different types of reversed-phase C18 chromatographic columns are used by using a phosphate buffer system and using 84/1/15v/v/v methanol/tetrahydrofuran/10 mM ammonium dihydrogen phosphate (pH3.0) as a mobile phase.
Example 2
Based on the "chinese pharmacopoeia" 2020 edition (second part), the present example uses a phosphate buffer system, the same mobile phase, and reversed phase C18 chromatographic columns from different manufacturers to perform the test, and the high performance liquid chromatogram is shown in fig. 2, and the test results are shown in table 2.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: single point bonding with end capping C18 (4.6X 250mm, 5 μm);
the imported brand Kromasil 100-5-C18 (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/10 mM ammonium dihydrogen phosphate, pH 3.0;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: control solution.
TABLE 2
As can be seen from Table 2, the reversed phase C18 chromatographic columns of different manufacturers, which use a phosphate buffer system and use 84/1/15v/v/v methanol/tetrahydrofuran/10 mM ammonium dihydrogen phosphate (pH3.0) as a mobile phase, have symmetrical peak shapes and high column efficiency during detection of the biphenyl benzyl oxazole (peak 1).
Example 3
Based on the "chinese pharmacopoeia" 2020 edition (second part), the present example uses an acetate buffer system, the same mobile phase, and different types of reverse phase C18 chromatographic columns for testing, and the high performance liquid chromatogram is shown in fig. 3, and the test results are shown in table 3.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: single point bonding with end capping C18 (4.6X 250mm, 5 μm);
non-end-capped C18 with single point linkage of side chain protection (4.6X 250mm, 5 μm);
multi-point bonding with end capping C18 (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/20 mM ammonium acetate, pH 5.2;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: control solution.
TABLE 3
As can be seen from Table 3, the peak shape of bifonazole (peak 1) is symmetrical and the column efficiency is high when three different types of reversed-phase C18 chromatographic columns are used by using an acetate buffer system and using 84/1/15v/v/v methanol/tetrahydrofuran/20 mM ammonium acetate (pH5.2) as a mobile phase.
Example 4
Based on the "chinese pharmacopoeia" 2020 edition (second part), the present example uses an acetate buffer system, the same mobile phase, and reversed phase C18 chromatographic columns from different manufacturers to perform the test, and the high performance liquid chromatogram is shown in fig. 4, and the test results are shown in table 4.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: single point bonding with end capping C18 (4.6X 250mm, 5 μm);
the imported brand Kromasil 100-5-C18 (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/20 mM ammonium acetate, pH 5.2;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: control solution.
TABLE 4
As shown in Table 4, the peak shape of biphenyl benzoazole (peak 1) is symmetrical and the column efficiency is high when the detection is carried out by using an acetate buffer system and using 84/1/15v/v/v methanol/tetrahydrofuran/10 mM ammonium acetate (pH5.2) as a mobile phase and different reversed-phase C18 chromatographic columns from factories.
Example 5
On the basis of the "chinese pharmacopoeia" 2020 edition (second part), under a considerable retention factor, the separation selectivity was examined by using an acetate buffer system and an acetate buffer system, respectively, and the high performance liquid chromatogram is shown in fig. 5, and the test results are shown in table 5.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: non-end-capped C18 with single point linkage of side chain protection (4.6X 250mm, 5 μm);
mobile phase: mobile phase A: 84/1/15v/v/v methanol/tetrahydrofuran/20 mM ammonium acetate, pH 5.2;
mobile phase B: 77/1/22v/v/v methanol/tetrahydrofuran/10 mM ammonium dihydrogen phosphate, pH 3.0;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: system suitability solution.
TABLE 5
As can be seen from table 5, the phosphate buffer system and the acetate buffer system show different separation selectivity at comparable retention factors; ammonium acetate buffer systems are preferred in the present invention, given their compatibility with mass spectrometry, which facilitates the identification of the relevant impurities.
Example 6
Based on the "chinese pharmacopoeia" 2020 edition (second part), this example uses an acetate buffer system, the same mobile phase, and different types of reverse phase C18 chromatographic columns for testing, and examines the separation selectivity of bifonazole and related impurities (including biphenyl benzyl alcohol), and the high performance liquid chromatogram is shown in fig. 6, and the test results are shown in table 6.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: single point bonding with end capping C18 (4.6X 250mm, 5 μm);
non-end-capped C18 with single point linkage of side chain protection (4.6X 250mm, 5 μm);
multi-point bonding with end capping C18 (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/20 mM ammonium acetate, pH 5.2;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: system suitability solution.
TABLE 6
As can be seen from fig. 6, the same mobile phase with the ammonium acetate buffer system, the C18 column with single point linkage side chain protection without end capping (chromatcore AR C18) performed best.
Example 7
Based on the "chinese pharmacopoeia" 2020 edition (second part), this example uses an acetate buffer system and a simplified mobile phase without adding tetrahydrofuran to perform a test, and examines the separation selectivity of bifonazole and related impurities (including biphenyl benzyl alcohol), and a high performance liquid chromatogram is shown in fig. 7, and the test results are shown in table 7.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: chromcore AR C18 (4.6X 250mm, 5 μm);
mobile phase: mobile phase A: 84/1/15v/v/v methanol/tetrahydrofuran/20 mM ammonium acetate, pH 5.2;
mobile phase B: 85/15v/v methanol/20 mM ammonium acetate, pH 5.2;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: system suitability solution (1 mg/mL).
TABLE 7
As can be seen from Table 7, the simplified mobile phase (without addition of tetrahydrofuran) shows comparable or slightly superior selectivity of separation, and the tetrahydrofuran-free mobile phase is preferred in the present invention.
Example 8
This example was tested using an acetate buffer system and a simplified mobile phase without tetrahydrofuran based on the "Chinese pharmacopoeia" 2020 edition (second division), and biphenyl benzyl alcohol (5. mu.g/mL) and biphenyl benzyl azole (10. mu.g/mL) control solutions (10 needles) were analyzed continuously, and the test results are shown in Table 8.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: chromcore AR C18 (4.6X 250mm, 5 μm);
mobile phase: 85/15v/v methanol/20 mM ammonium acetate, pH 5.2;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: biphenyl benzyl alcohol (5. mu.g/mL) + Biphenyl benzyl azole (10. mu.g/mL).
TABLE 8
As can be seen from Table 8, with the acetate buffer system, no tetrahydrofuran was added to the mobile phase, and the results of repeated experiments showed that the retention time and the peak area (signal response) were very good.
Example 9
This example considers the linear relationship, detection limit and quantification limit under the preferred conditions: the column was prepared using Chromcore AR C18 (4.6X 250mm, 5 μm); the mobile phase is 85/15v/v methanol/20 mM ammonium acetate, pH5.2; the regression equation and linear range of biphenyl benzyl alcohol and biphenyl benzyl azole are shown in fig. 8 and fig. 9.
Detection limit and quantitation limit: and respectively taking control solutions of the biphenyl benzyl alcohol and the biphenyl benzyl azole, gradually diluting, wherein when the signal to noise ratio is 3, the detection limits of the biphenyl benzyl alcohol and the biphenyl benzyl azole are respectively 3.3ng/mL and 15.3ng/mL, and when the signal to noise ratio is 10, the quantification limits of the biphenyl benzyl alcohol and the biphenyl benzyl azole are respectively 11.1ng/mL and 51.0 ng/mL.
Example 10
The applicability of the bifonazole system under the preferred conditions is tested by the embodiment, and the test requirements are as follows: in the system applicability solution chromatogram of biphenyl benzyl oxazole required by Chinese pharmacopoeia, the separation degree between the biphenyl benzyl oxazole peak and the biphenyl benzyl alcohol peak is required to meet the requirement, the high performance liquid chromatogram is shown in figure 10, and the test result is shown in table 9.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: chromcore AR C18 (4.6X 250mm, 5 μm);
mobile phase: 85/15v/v methanol/20 mM ammonium acetate, pH 5.2;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: system suitability solution (1 mg/mL).
TABLE 9
As can be seen from table 9, the degree of separation between the biphenyl oxazole peak and the biphenyl benzyl alcohol peak under the preferred conditions of the present invention is good, and has good system applicability.
Comparative example 1
On the basis of the 'Chinese pharmacopoeia' 2020 edition (second part), the comparative example uses the same mobile phase and adopts different types of chromatographic columns for testing, the high performance liquid chromatogram is shown in figure 11, and the test results are shown in table 10.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: single point bonding with end capping C18 (4.6X 250mm, 5 μm);
non-end-capped C18 with single point linkage of side chain protection (4.6X 250mm, 5 μm);
multi-point bonding with end capping C18 (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/water;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: control solution.
As can be seen from FIG. 11, the phenomena of asymmetric peak shape and poor peak shape of bifonazole can be seen after different types of chromatographic columns are tested by using 84/1/15v/v/v methanol/tetrahydrofuran/water as a mobile phase without adopting a buffer system.
Comparative example 2
On the basis of the 'Chinese pharmacopoeia' 2020 edition (second part), the comparative example uses the same mobile phase, different manufacturers and the same type of chromatographic columns for testing, the high performance liquid chromatogram is shown as figure 12, and the test results are shown as table 11.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: single point bonding with end capping C18 (4.6X 250mm, 5 μm);
imported brand K C18 (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/water;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: control solution.
TABLE 11
As can be seen from FIG. 12, the phenomena of asymmetric peak shape and poor peak shape of the bifonazole are generated after chromatographic column tests of different manufacturers by adopting 84/1/15v/v/v methanol/tetrahydrofuran/water as a mobile phase without adopting a buffer system.
Comparative example 3
On the basis of the "Chinese pharmacopoeia" 2020 edition (second part), the comparative example uses the same mobile phase and the same chromatographic column to continuously analyze a sample control solution (3 needles), the high performance liquid chromatogram is shown in FIG. 13, and the test results are shown in Table 12.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: non-end-capped C18 with single point linkage of side chain protection (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/water;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: control solution.
TABLE 12
As can be seen from Table 12 and FIG. 13, the sample control solution was continuously analyzed on the same column without using a buffer system and using 84/1/15v/v/v methanol/tetrahydrofuran/water as a mobile phase, and the retention time and the reproducibility of the peak shape were poor.
Comparative example 4
Based on the 'Chinese pharmacopoeia' 2020 edition (second part), the same mobile phase preparation method is used in the comparative example, but the mobile phase batches are different, the separation effect is tested on the same chromatographic column, the high performance liquid chromatogram is shown in fig. 14, and the test results are shown in table 13.
The detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: non-end-capped C18 with single point linkage of side chain protection (4.6X 250mm, 5 μm);
mobile phase: 84/1/15v/v/v methanol/tetrahydrofuran/water (batch A);
84/1/15v/v/v methanol/tetrahydrofuran/water (batch B);
84/1/15v/v/v methanol/tetrahydrofuran/water (batch C);
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: UV 254 nm;
sample preparation: control solution.
Watch 13
As can be seen from table 13 and fig. 14, 84/1/15v/v/v methanol/tetrahydrofuran/water was prepared as a mobile phase by the same preparation method without using a buffer system, and the retention time and peak shape of bifonazole were variable in different mobile phase batches by using the same chromatographic column.
According to the high performance liquid chromatography method for measuring related substances in the bifonazole bulk drug, the buffer solution is adopted to replace pure water, so that the peak shape, column efficiency and reproducibility of the bifonazole are obviously improved. Wherein the phosphate buffer system and the acetate buffer system show different separation selectivity; acetate buffer systems are preferred in the present invention, considering that they are compatible with mass spectrometry, facilitating the identification of the relevant impurities.
Based on the current Chinese pharmacopoeia method, under a 20mM ammonium acetate (pH5.2) buffer system, different types of C18 reverse phase chromatographic columns show different separation effects of bifonazole and related impurities (including biphenyl benzyl alcohol), and an uncapped C18 chromatographic column (Chromcore AR C18) with single-point bonded side chain protection is the best chromatographic column, which is the preferred chromatographic column in the invention.
Simplified mobile phases (without added tetrahydrofuran) show comparable or slightly superior selectivity of separation using ammonium acetate buffer systems, and therefore tetrahydrofuran-free mobile phase systems are preferred in the present invention.
Under the preferred chromatographic conditions (85/15 v/v methanol/20 mM ammonium acetate, pH5.2, mobile phase with non-blocked single-point bonded C18 with side chain protection as a chromatographic column, biphenyl benzyl alcohol (5 μ g/mL) and biphenyl benzyl azole (10 μ g/mL) as control solutions, the retention time RSD of biphenyl benzyl alcohol is 0.027%, the peak area RSD is 0.129%, the retention time RSD of biphenyl benzyl azole is 0.028%, the peak area RSD is 0.038%, and the reproducibility is good.
Under the preferred chromatographic conditions (85/15 v/v methanol/20 mM ammonium acetate as mobile phase, pH5.2) with the non-blocked single-point bonded C18 with side chain protection as the chromatographic column, the separation degree of biphenyl benzyl alcohol and biphenyl benzyl azole is 7.70, the concentration of biphenyl benzyl alcohol is in the range of 1.00-10.0 μ g/mL, the linear equation is 1.1639x-0.0147, and the correlation coefficient R is20.9999, the linear relation between the peak area and the concentration is good, the detection limit is 3.3ng/mL, and the quantification limit is 11.1 ng/mL; the concentration of the bifonazole is within the range of 2.00-20.0 mu g/mL, the linear equation is that y is 0.7789x-0.0898, and the correlation coefficient R20.9996 shows a good linear relationship between peak area and concentration, with a detection limit of 15.3ng/mL and a quantification limit of 51.0 ng/mL.
The present invention is illustrated by the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, i.e. it is not meant to imply that the present invention must rely on the above-mentioned detailed process equipment and process flow to be practiced. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A high performance liquid chromatography method for measuring related substances in a bifonazole raw material medicine is characterized by comprising the following steps:
1) dissolving a to-be-detected sample of the bifonazole bulk drug in a solvent, and filtering to obtain a to-be-detected solution;
2) the method comprises the steps of adopting a mixed solution of a buffer solution and a solvent as a mobile phase, measuring a standard solution and a solution to be measured by using a high performance liquid chromatography to obtain a standard curve, and analyzing the content of bifonazole and related substances in the solution to be measured according to the standard curve.
2. The high performance liquid chromatography method according to claim 1, wherein in step 2), the buffer in the mobile phase is an acetate buffer or a phosphate buffer, preferably an acetate buffer;
preferably, the solvent is an organic solvent;
preferably, the organic solvent is methanol.
3. The high performance liquid chromatography method according to claim 2, wherein in step 2), the mobile phase is a mixture of an acetate buffer and methanol;
preferably, the volume ratio of the acetate buffer to the methanol is (80-90): 10-20), preferably 85: 15.
4. The high performance liquid chromatography method of claim 2, wherein in step 2), the acetate in the acetate buffer comprises ammonium, sodium and potassium acetate salts and hydrates thereof.
5. The high performance liquid chromatography method according to any one of claims 1 to 4, wherein in step 2), the high performance liquid chromatography column is a reversed phase liquid chromatography column, preferably a C18 column.
6. The high performance liquid chromatography method according to claim 5, wherein the C18 column is an uncapped C18 column with single point bonding side chain protection, preferably a Chromcore AR C18 column.
7. The high performance liquid chromatography method according to any one of claims 1 to 6, wherein in step 2) the pH of the mobile phase is between 5 and 5.4, preferably 5.2.
8. The high performance liquid chromatography method according to any one of claims 1 to 7, wherein the detection conditions of the high performance liquid chromatography in step 2) are as follows: column temperature of the column: the flow rate of the mobile phase is 0.9-1.1mL/min at 20-30 ℃, and the detection wavelength is 256 nm.
9. The high performance liquid chromatography method according to one of claims 1 to 8, wherein in step 1), the solvent is a mixed solution of methanol and water in a volume ratio of (80-90) to (10-20), preferably a mixed solution of methanol and water in a volume ratio of 85: 15.
10. The high performance liquid chromatography method according to any one of claims 1 to 8, wherein the substance of interest in the bifonazole bulk drug comprises biphenyl benzyl alcohol.
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