CN111693627B - Detection method and application of 6-chloro-3-methyl uracil and related substances thereof - Google Patents
Detection method and application of 6-chloro-3-methyl uracil and related substances thereof Download PDFInfo
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Abstract
The invention relates to the technical field of chemical drug analysis methods, in particular to a detection method and application of 6-chloro-3-methyl uracil and related substances thereof. The detection method of 6-chloro-3-methyl uracil and its related substances includes the following steps: detecting the test solution by high performance liquid chromatography; the detection conditions of the high performance liquid chromatography comprise: the detection wavelength is 205-215 nm; performing isocratic elution by using a mobile phase, wherein the mobile phase comprises a mobile phase A and a mobile phase B at a volume ratio of 5: 95-15: 85, the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution; the mass fraction of the phosphoric acid aqueous solution is 0.05-0.3%. The detection method can realize one-time efficient separation of 6-chloro-3-methyl uracil and related substances thereof, has strong specificity and good durability, and can be used for research and quality control of 6-chloro-3-methyl uracil.
Description
Technical Field
The invention relates to the technical field of chemical drug analysis methods, in particular to a detection method and application of 6-chloro-3-methyl uracil and related substances thereof.
Background
6-chloro-3-methyl uracil is a key starting material for synthesizing trelagliptin succinate and alogliptin benzoate, and has the following structures:
in the process of synthesizing 6-chloro-3-methyl uracil, 1-methyl barbituric acid is obtained by malonic acid and methylurea under the action of acetic anhydride, and part of the 1-methyl barbituric acid may be left because the reaction is not complete; in addition, 1-methyl barbituric acid can generate 6-chloro-3-methyl uracil under the action of phosphorus oxychloride, and simultaneously generate 6-chloro uracil by demethylation. 1-methyl barbituric acid and 6-chlorouracil may participate in the reaction to produce impurity in the production process of raw material medicine, so that the strict control of the quality standard of 6-chloro-3-methyl uracil is of great importance to the quality and safety of medicine.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for detecting 6-chloro-3-methyl uracil and related substances thereof, which can ensure efficient separation of the related substances from the 6-chloro-3-methyl uracil, and has strong specificity and good durability.
The second purpose of the invention is to provide the application of the detection method of 6-chloro-3-methyl uracil and related substances thereof in the quality control of raw materials or preparations of 6-chloro-3-methyl uracil.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the detection method of 6-chloro-3-methyl uracil and its related substances includes the following steps:
detecting the test solution by high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
the detection wavelength is 205-215 nm;
performing isocratic elution by using a mobile phase, wherein the mobile phase comprises a mobile phase A and a mobile phase B in a volume ratio of 5: 95-15: 85, the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution;
the mass fraction of the phosphoric acid aqueous solution is 0.05-0.3%.
The detection method can realize one-time efficient separation of 6-chloro-3-methyl uracil and related substances thereof, has strong specificity and good durability, and can be used for research and quality control of 6-chloro-3-methyl uracil.
In a specific embodiment of the present invention, the related substances include I and II, which have the following structural formulas:
The related substances of the 6-chloro-3-methyl uracil may participate in the reaction in the production process of the raw material medicines to generate impurities, so that the detection method of the 6-chloro-3-methyl uracil and the related substances is of great importance to guarantee the product quality of the 6-chloro-3-methyl uracil.
The detection method can separate and detect the 6-chloro-3-methyluracil, the 1-methylbarbituric acid and the 6-chlorouracil at one time and high efficiency.
As in the different embodiments of the present invention, the detection wavelength may be 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, and so on. Preferably, the detection wavelength is 208-212 nm, and more preferably 210 nm.
By adopting the detection wavelength, the separation degree of the 6-chloro-3-methyluracil and related substances thereof can be further improved, and the peak area difference of the 6-chloro-3-methyluracil, the impurity I and the impurity II is small.
As in the different embodiments of the invention, the volume ratio of mobile phase a to mobile phase B in the mobile phase can be 5: 95, 6: 94, 7: 93, 8: 92, 9: 91, 10: 90, 11: 89, 12: 88, 13: 87, 14: 86, 15: 85, etc. Preferably, the volume ratio of the mobile phase A to the mobile phase B in the mobile phase is 8: 92 to 12: 88, and more preferably 10: 90.
By adopting the mobile phase proportion, the peak-appearing time interval of the 6-chloro-3-methyluracil, the impurity I and the impurity II is longer, and the separation degree is better.
As in the various embodiments of the present invention, the mass fraction of the phosphoric acid aqueous solution may be 0.05%, 0.08%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, etc. Preferably, the phosphoric acid aqueous solution is 0.08 to 0.15% by mass, more preferably 0.1% by mass.
In a specific embodiment of the invention, the chromatographic column of the high performance liquid chromatography is an octadecylsilane bonded silica chromatographic column. Specifically, octadecylsilane bonded silica (250X 4.6mm, 5 μm or equivalent) may be used as the filler. 150X 4.6mm, 5 μm octadecylsilane chemically bonded silica may also be used as a filler.
In a specific embodiment of the invention, the column temperature of the chromatographic column is 25-45 ℃. As in the different embodiments, the column temperature of the chromatographic column can be 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃ and so on. Preferably, the temperature of the chromatographic column is 30-40 ℃, and more preferably 35 ℃.
In a specific embodiment of the invention, the flow rate of the isocratic elution is 0.8-1.2 mL/min. As in various embodiments, the flow rate of the isocratic elution can be 0.8mL/min, 0.85mL/min, 0.9mL/min, 0.95mL/min, 1.0mL/min, 1.05mL/min, 1.1mL/min, 1.15mL/min, 1.2mL/min, and the like. Preferably, the flow rate of the isocratic elution is 0.9-1.1 mL/min, and more preferably 1.0 mL/min.
In an embodiment of the present invention, the sample amount of the sample solution may be 1 to 10 μ L, for example, 5 μ L.
In a specific embodiment of the present invention, the method for preparing the test solution comprises: and dissolving the sample by using the mobile phase A, and diluting by using the mobile phase. The specific dilution factor is adjusted according to actual requirements, and preferably, the concentration of 6-chloro-3-methyluracil in the sample solution is 0.1-1.0 mg/mL, for example, about 0.5 mg/mL. In the specific operation, the concentration of the main component 6-chloro-3-methyl uracil in the sample solution is not limited to this, as long as the concentration x the sample injection volume > the limit of quantification of each impurity is satisfied. Specifically, the sample solution with the concentration of 0.5 +/-0.01 mg/mL is prepared by precisely weighing 25mg +/-0.5 mg of the sample, placing the sample in a 50mL measuring flask, adding the mobile phase A5mL for dissolution, and diluting the sample solution to a scale by adopting the mobile phase.
In a specific embodiment of the invention, the test article comprises a 6-chloro-3-methyluracil-containing material or formulation.
In a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography include:
detection wavelength: 210 nm;
isocratic elution is carried out by adopting a mobile phase, wherein the mobile phase comprises acetonitrile and a phosphoric acid aqueous solution in a volume ratio of 10: 90, and the mass fraction of the phosphoric acid aqueous solution is 0.1%;
the flow rate of isocratic elution is 1.0 mL/min;
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column with the specification of 250 multiplied by 4.6mm and 5 mu m; the column temperature of the column was 35 ℃.
The detection method has the advantages that the elution time is within 25min, and the detection efficiency is higher under the condition of ensuring the effective separation of the main components and the related substances.
In a preferred embodiment of the present invention, the content of 6-chloro-3-methyluracil and/or related substances in the test sample solution is calculated by an external standard method.
Specifically, the method for calculating the content of 6-chloro-3-methyluracil comprises the following steps: respectively injecting 6-chloro-3-methyluracil standard series working solutions into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas under the detection conditions of the high performance liquid chromatograph, and drawing a 6-chloro-3-methyluracil standard curve by taking the concentration of the standard working solution as a horizontal coordinate and the peak area as a vertical coordinate; substituting the chromatographic peak area of the 6-chloro-3-methyl uracil in the chromatographic detection result of the test solution into the 6-chloro-3-methyl uracil standard curve, and calculating to obtain the concentration of the 6-chloro-3-methyl uracil in the test solution. Wherein, the concentration range of the 6-chloro-3-methyl uracil standard series working solution is 0.5-55 μ g/mL, for example, working solutions with concentrations of about 0.5 μ g/mL, 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL, etc. can be specifically adopted.
Specifically, the method for calculating the content of the impurity I comprises the following steps: respectively injecting standard series working solutions of impurities I into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas under the detection condition of the high performance liquid chromatograph, and drawing a standard curve of the impurities I by taking the concentration of the standard working solutions as a horizontal coordinate and the peak areas as a vertical coordinate; substituting the chromatographic peak area of the impurity I in the chromatographic detection result of the test solution into the standard curve of the impurity I, and calculating to obtain the concentration of the impurity I in the test solution. The concentration range of the impurity I standard series working solution is 0.5-55 mu g/mL, and for example, the working solution with the concentration of about 0.5 mu g/mL, 1 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL, 50 mu g/mL and the like can be specifically adopted.
Specifically, the method for calculating the content of the impurity II comprises the following steps: respectively injecting standard series working solutions of impurities II into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas under the detection conditions of the high performance liquid chromatograph, and drawing an impurity II standard curve by taking the concentration of the standard working solutions as a horizontal coordinate and the peak areas as a vertical coordinate; substituting the chromatographic peak area of the impurity II in the chromatographic detection result of the test solution into the impurity II standard curve, and calculating to obtain the concentration of the impurity II in the test solution. The concentration range of the impurity II standard series working solution is 0.5-55 mu g/mL, and for example, working solutions with concentrations of about 0.5 mu g/mL, 1 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL, 50 mu g/mL and the like can be specifically adopted.
In the preparation of the standard series working solution, the mobile phase A is adopted to dissolve the corresponding reference substance, and the mobile phase is adopted to dilute the reference substance to the preset concentration.
The invention also provides the application of the detection method of the 6-chloro-3-methyl uracil and related substances thereof in the quality control of raw materials or preparations of the 6-chloro-3-methyl uracil.
The detection method can realize one-time efficient separation of 6-chloro-3-methyl uracil and related substances thereof, and has the advantages of strong specificity, good durability, high sensitivity, good repeatability, good linear relation, good accuracy, good solution stability and the like on the basis of ensuring the efficient separation of the 6-chloro-3-methyl uracil and the related substances.
Compared with the prior art, the invention has the beneficial effects that:
(1) the detection method can realize one-time efficient separation of 6-chloro-3-methyl uracil and related substances thereof, has strong specificity and good durability, and can be used for research and quality control of 6-chloro-3-methyl uracil;
(2) the detection method of the invention can be used for the quality control of the raw material or preparation of the 6-chloro-3-methyl uracil.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the linear relationship between 6-chloro-3-methyluracil provided in example 3 of the present invention;
FIG. 2 is a linear relationship of impurity I provided in example 4 of the present invention;
FIG. 3 is a linear relationship diagram of impurity II provided in example 5 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the specific implementation mode, the verification of the items such as specificity, detection limit and quantification limit, precision, linearity and range, accuracy, solution stability and the like is performed according to the technical guide principle of verification of chemical drug quality control analysis method, the technical guide principle of standardized process established by chemical drug quality standard, the technical guide principle of chemical drug impurity research, the technical guide principle of chemical drug residual solvent research and the related guide principle in the appendix of the current edition of the pharmacopoeia of the people's republic of China.
Example 1
This example provides a method for detecting 6-chloro-3-methyluracil and related substances, comprising the following steps:
(1) test solution
Preparation of a test solution: precisely weighing 25.17mg of 6-chloro-3-methyluracil, placing the 6-chloro-3-methyluracil in a 50mL volumetric flask, adding 5mL of acetonitrile, precisely weighing 0.5mL of impurity I solution and 0.5mL of impurity II solution in the volumetric flask, dissolving and diluting the impurity I solution and the impurity II solution to a scale by using a mobile phase, and shaking up to obtain the product;
the preparation method of the impurity I solution comprises the following steps: accurately weighing 25.12mg of impurity I, placing the impurity I in a 50mL volumetric flask, adding 5mL of acetonitrile to dissolve the impurity I, diluting the impurity I to a scale by using a mobile phase, and shaking up the impurity I to obtain the product; the preparation method of the impurity II solution comprises the following steps: accurately weighing 25.13mg of impurity II, placing the impurity II in a 50mL volumetric flask, adding 5mL of acetonitrile to dissolve the impurity II, diluting the impurity II to a scale by using a mobile phase, and shaking up the impurity II to obtain the product.
(2) High performance liquid chromatography detection conditions
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, ultraviolet detector, detection wavelength: 210 nm;
a chromatographic column: agilent SB C18(150 mm. times.4.6 mm, 5 μm) column;
mobile phase: mobile phase a and mobile phase B; the volume ratio of the mobile phase A to the mobile phase B is 10: 90, and isocratic elution is carried out;
wherein the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with the mass fraction of 0.1%;
flow rate: 1.0 mL/min;
column temperature: 35 ℃;
sample introduction volume: 5 μ L.
(3) Detection step
Precisely measuring 5 mu L of the test solution, injecting into a liquid chromatograph, detecting according to the conditions in the step (2), and recording a chromatogram. In the chromatogram, each peak is sequentially impurity I, impurity II and 6-chloro-3-methyluracil, the retention time is respectively 2.985min, 3.636min and 7.346min, and the separation degree among the impurities and between the impurities and the main component is good.
Comparative example 1: referring to the detection conditions of example 1, the volume ratio of mobile phase a to mobile phase B was changed to 25: 75, and the remaining conditions were not changed, and the retention times are shown in table 1.
TABLE 16 retention times of chloro-3-methyluracil and related substances at different mobile phase ratios
As can be seen from Table 1, when the volume ratio of the mobile phase A to the mobile phase B was 10: 90, the peak appearance time interval of 6-chloro-3-methyluracil, impurity I and impurity II was long, and 6-chloro-3-methyluracil and related substances could be efficiently separated and detected.
Comparative example 2: referring to the detection conditions of example 1, the detection wavelength was changed to 261nm, and the remaining conditions were not changed, and the peak areas of 6-chloro-3-methyluracil, impurity I and impurity II are shown in Table 2.
TABLE 26 areas of peak of chloro-3-methyluracil and related substances at different detection wavelengths
As is clear from Table 2, the difference in peak area between 6-chloro-3-methyluracil, impurity I and impurity II at the wavelength of 210nm is small, and the preferable wavelength is obtained.
Example 2
This example refers to the detection method of example 1, with the only difference that: in the step (2), the chromatographic column is a C18(250 mm. times.4.6 mm, 5 μm) chromatographic column, the remaining detection conditions are unchanged, and the retention times of 6-chloro-3-methyluracil, impurity I and impurity II are specifically shown in Table 3.
TABLE 36 Retention time of chloro-3-methyluracil and related substances under different column conditions
As is clear from Table 3, the column having a size of 4.6X 250mm and 5 μm was used, and the intervals between the peaks of 6-chloro-3-methyluracil, impurity I and impurity II were long, and the degree of separation was more excellent, and the column having a size of 250mm X4.6 mm and 5 μm was suitable.
Example 3
This example provides a method for detecting the content of 6-chloro-3-methyluracil, which includes the following steps:
(1) precisely weighing 2.510mg of 6-chloro-3-methyluracil, placing the 6-chloro-3-methyluracil in a 50mL vial, dissolving the 6-chloro-3-methyluracil in 5mL of acetonitrile, quantitatively diluting the 6-chloro-3-methyluracil with a mobile phase (acetonitrile: 0.1 wt% phosphoric acid aqueous solution ═ 10: 90) to obtain mother solutions (linear 6), measuring the mother solutions in 1mL, and 2mL to 100mL (linear 1), 50mL (linear 2), 10mL (linear 3), 5mL (linear 4), and 5mL (linear 5) vials, and quantitatively diluting the mother solutions in the mobile phase (acetonitrile: 0.1 wt% phosphoric acid aqueous solution ═ 10: 90) to obtain 6-chloro-3-methyluracil standard series working solutions having the concentrations shown in table 4;
(2) precisely measuring 5 μ L of each of the standard series working solutions, injecting into a liquid chromatograph, detecting according to the detection conditions in the embodiment 2, and recording the chromatogram; the test results are shown in Table 4, and a linear regression equation is obtained by using the concentration C (μ g/mL) as the abscissa and the corresponding peak area as the ordinate, and the linear relationship is shown in FIG. 1.
The linear relation between the concentration of the 6-chloro-3-methyl uracil and the peak area is good, and the verification requirement is met.
TABLE 46 Linear test results for chloro-3-methyluracil
Example 4
The embodiment provides a method for detecting the content of an impurity I, which comprises the following steps:
(1) precisely weighing 2.503mg of impurity i, dissolving the impurity i in 5mL of acetonitrile, quantitatively diluting the solution to a mother solution (linear 6) in a scale by using a mobile phase (acetonitrile: 0.1 wt% phosphoric acid aqueous solution ═ 10: 90), weighing the mother solutions 1mL, 2mL to 100mL (linear 1), 50mL (linear 2), 10mL (linear 3), 5mL (linear 4) and 5mL (linear 5) in vials, and quantitatively diluting the solutions to impurity i standard series working solutions with the concentrations shown in table 5 by using the mobile phase (acetonitrile: 0.1 wt% phosphoric acid aqueous solution ═ 10: 90);
(2) precisely measuring 5 μ L of each of the standard series working solutions, injecting into a liquid chromatograph, detecting according to the detection conditions in the embodiment 2, and recording the chromatogram; the test results are shown in Table 5, and a linear regression equation is obtained by using the concentration C (μ g/mL) as the abscissa and the corresponding peak area as the ordinate, and the linear relationship is shown in FIG. 2.
The linear relation between the concentration of the impurity I and the peak area is good, and the verification requirement is met.
TABLE 5 Linear test results for impurity I
Example 5
The embodiment provides a method for detecting the content of an impurity II, which comprises the following steps:
(1) 2.506mg of impurity ii was precisely weighed out, dissolved in 5mL of acetonitrile, and quantitatively diluted with a mobile phase (acetonitrile: 0.1 wt% phosphoric acid aqueous solution ═ 10: 90) to give a mother liquor (linear 6), and in vials of 1mL, and 2mL to 100mL (linear 1), 50mL (linear 2), 10mL (linear 3), 5mL (linear 4), and 5mL (linear 5), respectively, quantitatively diluted with a mobile phase (acetonitrile: 0.1 wt% phosphoric acid aqueous solution ═ 10: 90) to give a standard series of working solutions of impurity ii having the concentrations shown in table 6;
(2) precisely measuring 5 μ L of each of the standard series working solutions, injecting into a liquid chromatograph, detecting according to the detection conditions in the embodiment 2, and recording the chromatogram; the test results are shown in Table 6, and a linear regression equation is obtained by using the concentration C (μ g/mL) as the abscissa and the corresponding peak area as the ordinate, and the linear relationship is shown in FIG. 3.
The linear relation between the concentration of the impurity II and the peak area is good, and the verification requirement is met.
TABLE 6 Linear test results for impurity II
Experimental example 1
Specificity
(1) Blank reagent interference test
The mobile phase (5. mu.L) was precisely measured, and the sample was injected into a liquid chromatograph under the chromatography conditions in example 2, and the chromatogram was recorded. And as can be seen from the chromatogram, the blank solvent is not interfered.
Wherein the mobile phase is a mixed solution of acetonitrile and 0.1% phosphoric acid aqueous solution at a volume ratio of 10: 90.
(2) System suitability test
Test solution: accurately weighing 24.99mg of 6-chloro-3-methyluracil, placing the 6-chloro-3-methyluracil in a 50mL volumetric flask, adding 5mL of acetonitrile to dissolve the 6-chloro-3-methyluracil, diluting the solution to a scale by using a mobile phase, and shaking up the solution to obtain the compound.
Solution of impurity I: accurately weighing 25.12mg of impurity I, placing the impurity I in a 50mL volumetric flask, adding 5mL of acetonitrile to dissolve the impurity I, diluting the impurity I to a scale with a mobile phase, and shaking up to obtain the product.
Impurity II solution: accurately weighing 25.13mg of impurity II, placing the impurity II in a 50mL volumetric flask, adding 5mL of acetonitrile to dissolve the impurity II, diluting the impurity II to a scale by using a mobile phase, and shaking up the impurity II to obtain the product.
Mixing the solution: precisely weighing 25.17mg of 6-chloro-3-methyluracil, placing the 6-chloro-3-methyluracil in a 50mL volumetric flask, adding 5mL of acetonitrile, precisely weighing 0.5mL of each of the impurity solution I and the impurity solution II in the volumetric flask, dissolving and diluting the impurity solution I and the impurity solution II to a scale by using a mobile phase, and shaking up to obtain the product.
The sample solution, the impurity solution I, the impurity solution II and the mixed solution were measured precisely at 5. mu.L each, and the solutions were injected into a liquid chromatograph under the chromatographic conditions in example 2, and the chromatogram was recorded.
As a result: the theoretical plate number of the 6-chloro-3-methyl uracil peak is not less than 2000; the separation degrees of the 6-chloro-3-methyl uracil, the impurity I and the impurity II meet the requirement.
Experimental example 2
Detection limit and quantification limit
Precisely weighing reference substances of 6-chloro-3-methyluracil, the impurity I and the impurity II respectively, dissolving the reference substances into acetonitrile to serve as stock solutions of the 6-chloro-3-methyluracil, the impurity I and the impurity II respectively, and gradually diluting the reference substances by adopting a mobile phase (acetonitrile and 0.1 wt% phosphoric acid aqueous solution in a volume ratio of 10: 90) to prepare a quantitative limit solution and a detection limit solution; the sample injection detection is carried out according to the chromatographic detection conditions in the example 2, the peak height which is three times of the baseline noise is taken as the detection limit, and the peak height which is ten times of the baseline noise is taken as the quantification limit.
The specific process of diluting the quantitative limiting solution and the test results, and the process of diluting the testing limiting solution and the test results are shown in tables 7 and 8, respectively.
TABLE 7 dilution procedure of quantitative limiting solution and test results
Wherein, 6-chloro-3-methyl uracil is taken as an example to explain the dilution method, 1mL → 100mL, 1mL → 10mL means that 1mL of stock solution of 6-chloro-3-methyl uracil with the initial concentration of 0.4998mg/mL is taken and diluted to 100mL by adopting a mobile phase; then 1mL of the diluted solution is taken and diluted to 10mL by adopting a mobile phase to obtain a limit solution for quantification. The process of limiting solution dilution in Table 8 below is the same.
TABLE 8 dilution procedure of detection limiting solution and detection results
The detection results show that the detection limit and the quantitative limit of the 6-chloro-3-methyluracil, the impurity I and the impurity II can meet the requirement of the detection sensitivity of the 6-chloro-3-methyluracil related substances.
Experimental example 3
Precision degree
(1) Method of producing a composite material
Samples of the same lot were sampled, and 6 parts of the mixed solution were prepared in parallel as a test solution with reference to the method for preparing the mixed solution in the specificity test of Experimental example 1.6 parts of test solution 5. mu.L was measured precisely, and the measured solution was injected into a liquid chromatograph under the chromatographic detection conditions in example 2, and the chromatogram was recorded and calculated by the area normalization method, and the calculation results are shown in Table 9.
TABLE 9 precision
From the results in table 9, it can be seen that the RSD value of the sample of the same lot is less than 2.0% after 6 parallel determinations, indicating that the detection method has good repeatability.
Experimental example 4
Accuracy of
(1) Method of producing a composite material
Preparing an impurity I reference substance solution: precisely measuring 0.5mL of mother liquor of impurity I under the linear detection item in example 4, placing the mother liquor in a 50mL volumetric flask, diluting the mother liquor to a scale by using a mobile phase (acetonitrile and 0.1 wt% phosphoric acid aqueous solution in a volume ratio of 10: 90), and shaking uniformly to obtain the product.
Preparing an impurity II reference substance solution: precisely measuring 0.5mL of mother liquor of the impurity II under the linear detection item in the example 5, placing the mother liquor in a 50mL volumetric flask, diluting the mother liquor to scales by using a mobile phase (acetonitrile and 0.1 wt% phosphoric acid aqueous solution in a volume ratio of 10: 90), and shaking uniformly to obtain the product.
Preparing a test solution: precisely weighing 25.06mg, 25.07mg and 25.00mg of 6-chloro-3-methyluracil, respectively placing the 6-chloro-3-methyluracil in 3 50mL volumetric flasks, adding 5mL of acetonitrile, respectively adding 0.4mL, 0.5mL and 0.6mL of mother liquor of impurity I and impurity II, respectively, diluting to a scale with a mobile phase (acetonitrile and 0.1 wt% phosphoric acid aqueous solution at a volume ratio of 10: 90), and shaking uniformly to obtain a solution with a concentration of 80%, a concentration of 100% and a concentration of 120%. The injection was repeated 3 times for each concentration.
The control solution and the sample solution were measured accurately at 5. mu.L each, and the solutions were measured by injecting into a liquid chromatograph under the chromatographic conditions in example 2.
(2) Results
TABLE 10 impurity I recovery results
TABLE 11 results for recovery of impurity II
The result shows that the average recovery rate of the impurities I and II is within the range of 98.0-102.0 percent; RSD is less than 2.0%, and accuracy is good.
Experimental example 5
Stability of solution
(1) Method of producing a composite material
Precisely weighing 25.08mg of 6-chloro-3-methyluracil, placing the weighed 6-chloro-3-methyluracil in a 50mL volumetric flask, adding 5mL of acetonitrile, 5mL of the mother solution of impurity I in example 4 and 5mL of the mother solution of impurity II in example 5, diluting the mixture to a scale with mobile phases (acetonitrile and 0.1 wt% phosphoric acid aqueous solution in a volume ratio of 10: 90), and shaking the mixture to obtain a sample solution. After standing at room temperature for 12 hours, 5. mu.L of the sample solution was measured precisely at the end of 0, 2, 4, 6, 8, 10, and 12 hours, and the sample solution was measured by injecting into a liquid chromatograph under the conditions of chromatography in example 2, and the stability of the sample solution after standing at room temperature for 12 hours was examined.
(2) Results
TABLE 12 examination of solution stability
The test results show that the RSD values are less than 2.0%, which indicates that the test solution is stable within 12 hours at room temperature.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (18)
- The method for detecting the 6-chloro-3-methyl uracil and related substances thereof is characterized by comprising the following steps of:detecting the test solution by high performance liquid chromatography;the detection conditions of the high performance liquid chromatography comprise:the detection wavelength is 205-215 nm;the chromatographic column of the high performance liquid chromatography is an octadecylsilane chemically bonded silica chromatographic column;performing isocratic elution by using a mobile phase, wherein the mobile phase comprises a mobile phase A and a mobile phase B in a volume ratio of 5: 95-10: 90, the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution;the mass fraction of the phosphoric acid aqueous solution is 0.05-0.3%;the related substances comprise I and II, and the structural formulas are respectively as follows:
- 2. the detection method according to claim 1, wherein the detection wavelength is 208 to 212 nm.
- 3. The detection method according to claim 1, wherein the detection wavelength is 210 nm.
- 4. The detection method according to claim 1, wherein the volume ratio of the mobile phase A to the mobile phase B in the mobile phase is 8: 92 to 10: 90.
- 5. The detection method according to claim 1, wherein the volume ratio of the mobile phase A to the mobile phase B is 10: 90.
- 6. The detection method according to claim 1, wherein the phosphoric acid aqueous solution is present in an amount of 0.08 to 0.15% by mass.
- 7. The detection method according to claim 6, wherein the mass fraction of the phosphoric acid aqueous solution is 0.1%.
- 8. The detection method according to claim 1, wherein the column temperature of the chromatography column is 25 to 45 ℃.
- 9. The detection method according to claim 8, wherein the column temperature of the chromatography column is 35 ℃.
- 10. The detection method according to claim 1, wherein the flow rate of the isocratic elution is 0.8 to 1.2 mL/min.
- 11. The detection method according to claim 1, wherein the sample volume of the sample solution is 1 to 10. mu.L.
- 12. The method according to claim 11, wherein the sample amount of the sample solution is 5. mu.L.
- 13. The detection method according to claim 1, wherein the content of 6-chloro-3-methyluracil and/or related substances in the test sample solution is calculated by an external standard method.
- 14. The method according to claim 13, wherein the calculation of the content of 6-chloro-3-methyluracil and/or related substances comprises: respectively injecting standard series working solutions of 6-chloro-3-methyluracil and/or related substances into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas under the detection condition of the high performance liquid chromatograph, and drawing a standard curve of the 6-chloro-3-methyluracil and/or related substances by taking the concentration of the standard working solutions as a horizontal ordinate and the peak areas as a vertical coordinate; substituting the chromatographic peak area of the 6-chloro-3-methyl uracil and/or related substances in the chromatographic detection result of the test solution into the standard curve of the 6-chloro-3-methyl uracil and/or related substances, and calculating to obtain the concentration of the 6-chloro-3-methyl uracil and/or related substances in the test solution.
- 15. The detection method according to claim 1, wherein the sample comprises a raw material or a preparation containing 6-chloro-3-methyluracil.
- 16. The detection method according to claim 15, wherein the preparation method of the test solution comprises: and dissolving the sample by using the mobile phase A, and diluting by using the mobile phase A.
- 17. The detection method according to claim 1, wherein the detection conditions of the high performance liquid chromatography comprise:the detection wavelength is 210 nm;isocratic elution is carried out by adopting a mobile phase, wherein the mobile phase comprises acetonitrile and a phosphoric acid aqueous solution in a volume ratio of 10: 90, and the mass fraction of the phosphoric acid aqueous solution is 0.1%;the flow rate of isocratic elution is 1.0 mL/min;the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column with the specification of 250 multiplied by 4.6mm and 5 mu m; the column temperature of the chromatographic column was 35 ℃.
- 18. Use of the method for detecting 6-chloro-3-methyluracil and related substances according to any one of claims 1 to 17 for quality control of a raw material or preparation containing 6-chloro-3-methyluracil.
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| CN105699547A (en) * | 2016-04-22 | 2016-06-22 | 中山万汉医药科技有限公司 | Method for measuring related substances in succinic acid Trelagliptin raw materials |
| CN108586360A (en) * | 2018-06-13 | 2018-09-28 | 峨眉山宏昇药业股份有限公司 | A kind of preparation method of the chloro- 3- methyluracils of 6- |
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