CN114264765A - Analysis method for determining related substances in glimepiride intermediate by using HPLC - Google Patents
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Abstract
The invention discloses an analytical method for determining related substances in a glimepiride intermediate by using HPLC (high performance liquid chromatography), and particularly relates to an HPLC analytical method for the glimepiride intermediate and 4 impurities in the glimepiride intermediate. The chromatographic column type, specification, detection wavelength, mobile phase type, mobile phase proportion, running time and the like in the method have the advantages that the impurities in the glimepiride intermediate can be quickly, simply, conveniently, accurately and efficiently eluted, separated and quantitatively detected, the impurity peak and the main peak can be completely separated, and the optimal detection result can be achieved; the analysis method is convenient to operate, short in running time, strong in specificity, high in sensitivity and stability, has a good linear curve in a low concentration range, has strong repeatability and accuracy, is not influenced by personnel and instruments, is stable and reliable, and provides a basis for research and development and quality detection of the compounds.
Description
Technical Field
The invention belongs to the field of chemical drug analysis method development, and particularly relates to an analysis method of related substances in a glimepiride intermediate.
Background
4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-carboxamido) ethyl ] benzenesulfonamide, CAS: 119018-29-0, having the formula:
the molecular formula is as follows: c16H21N3O4S molecular weight: 351.47
Glimepiride is a sulfonylurea antidiabetic agent, and is suitable for type 2 diabetes mellitus in which diet control, exercise therapy and weight reduction cannot sufficiently control blood sugar. The product was first developed by Hoechst Marion Roussel in germany in the early eighties, and was first marketed in germany and then subsequently marketed in several countries such as switzerland, sweden, danish. Approved by the U.S. Instrument and drug administration (FDA) in 1995, and marketed in China in 2000. Glimepiride, as a third-generation sulfonylurea hypoglycemic agent, has the advantages of strong hypoglycemic effect, small dosage, long action time, small side effect, safety, reliability and the like compared with other sulfonylurea drugs, is widely used and has high market demand. 4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-formamido) ethyl ] benzenesulfonamide is one of the intermediates of glimepiride in the international market at present.
In order to ensure the safety and effectiveness of the medicine, the quality of the medicine needs to be comprehensively considered, related substances are closely related to the quality, safety and curative effect of the medicine, and the existence of the related substances may reduce the curative effect of the medicine and even cause toxic and side effects, so that the related substances in the medicine need to be researched, detected and monitored to ensure the quality of the medicine. 4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-formamido) ethyl ] benzene sulfonamide is a key intermediate material, so that the research on the chromatographic conditions of the related substance detection method is of great significance.
In the prior art, impurity spectrum research on a glimepiride intermediate is not carried out, impurity analysis and methodology research on known impurities are not carried out, and only the chromatographic purity of the glimepiride intermediate is simply detected. There are no 4 known impurity locations and no impurity correction factors calculated in the chromatographic purity measurements. Therefore, an analysis method for detecting related substances in the glimepiride intermediate 4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-formamido) ethyl ] benzenesulfonamide is developed.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides an analysis method of related substances in a glimepiride intermediate, which has the advantages of high impurity separation degree, more detected impurities and high sensitivity, and can comprehensively, quickly, accurately and effectively monitor the quality level of the glimepiride intermediate.
The terms "glimepiride intermediate", "benzenesulfonamide" or "4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-carboxamido) ethyl ] benzenesulfonamide" in the present invention mean "4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-carboxamido) ethyl ] benzenesulfonamide".
In view of the above technical problems, the present inventors have made extensive and intensive studies to propose the following technical solutions: an analytical method for determining related substances in a glimepiride intermediate by using HPLC (high performance liquid chromatography):
stationary phase: octadecylsilane chemically bonded silica is used as a stationary phase;
mobile phase: the volume ratio of the phosphate buffer solution to the organic phase is (50-70): (30-50) the formed mixed solvent is a mobile phase;
column temperature: 20-30 ℃;
flow rate: 0.4-1.0 ml/min;
detection wavelength: 225 nm.
In one embodiment of the invention, the sample preparation is to dilute the sample with a mobile phase to prepare a solution of 0.05-0.2 mg/ml.
The glimepiride intermediate is 4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-formamido) ethyl ] benzenesulfonamide, and related substances are as follows:
impurity A: 3-ethyl-4-methyl-3-pyrrolin-2-one;
impurity B: 3- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) -ethyl ] -benzenesulfonamide;
impurity C: 2- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) -ethyl ] -benzenesulfonamide;
impurity D: 3-ethyl-4-methyl-2-oxo-3-pyrroline-N- (2-phenylethyl) carboxamide;
the structural formula of the impurities is as follows:
in some embodiments of the invention, the length of the chromatographic column adopted by the invention is 150-250 mm, and the preferred length is 150 mm; the diameter is 4-5 mm, and the preferred diameter is 4.6 mm; the filler particle size is 2.7-5.0 μm, and the optimized particle size is 2.7 μm. Further preferably, the chromatography column is: agilent Poroshell 120PFP pentafluorophenyl column or equivalent column (150mm 4.6mm,2.7 μm). Chromatographic columns manufactured by different manufacturers have different models, and a person skilled in the art can screen the chromatographic columns according to the models of the chromatographic columns.
In one embodiment of the present invention, the organic phase used in the present invention is methanol or acetonitrile, more preferably, the organic phase is acetonitrile.
In one embodiment of the present invention, the phosphate buffer solution is ammonium dihydrogen phosphate buffer solution. In some embodiments, the concentration of the ammonium dihydrogen phosphate buffer solution is 0.01-0.1 mol/L; in some embodiments, the ammonium dihydrogen phosphate buffer solution has a pH of from 3.0 to 5.0.
In one embodiment of the invention, the mobile phase adopted by the invention is ammonium dihydrogen phosphate buffer solution and acetonitrile, wherein the volume ratio of the ammonium dihydrogen phosphate buffer solution to the acetonitrile is 50-70: 30-50, and further preferably, the volume ratio of the ammonium dihydrogen phosphate buffer solution to the acetonitrile is 60: 40.
in some embodiments of the invention, the elution time is 25 to 60min, preferably 30 min.
In one embodiment of the invention, the flow rate of the mobile phase is preferably 0.5 ml/min.
In one embodiment of the invention, the column temperature is preferably 25 ℃.
In some embodiments of the invention, the sample amount is 10-20 μ l; in a specific embodiment of the invention, the sample size is preferably 20. mu.l.
In one embodiment of the invention, the method is subjected to specificity test, and the specific steps of isocratic elution by using a mobile phase are as follows: respectively preparing a system applicability solution, a test mixed solution, a glimepiride intermediate control solution and a glimepiride intermediate test solution, positioning and injecting the solutions of all the components, and calculating the content of each impurity according to an area normalization method.
In one embodiment, the specific steps for isocratic elution using a mobile phase are: injecting blank mobile phase.
In one embodiment, the analytical method is subjected to detection limit and quantification limit tests, and the specific steps of isocratic elution by using a mobile phase are as follows: and respectively preparing a glimepiride intermediate, a detection limit solution and a quantitative limit solution of each impurity, injecting samples respectively, and calculating a detection limit and a quantitative limit.
In one embodiment, the assay is subjected to both linear and range tests, and the specific steps of isocratic elution using a mobile phase are: and respectively preparing the glimepiride intermediate and linear solutions (the quantitative limit concentration is 120 percent of the limit concentration) with different concentrations of each impurity, respectively injecting samples, and calculating a linear equation and a linear range. In one example, the analytical method is subjected to repeated and intermediate precision tests, and the specific steps of isocratic elution using a mobile phase are as follows: and preparing a repetitive solution and an intermediate precision solution, injecting samples respectively, and calculating the RSD of each solution.
In one embodiment, the analytical method is tested for accuracy, and the specific steps of isocratic elution using a mobile phase are as follows: respectively preparing 20% sample adding and recovering solution, 50% sample adding and recovering solution and 120% sample adding and recovering solution, respectively injecting samples, and calculating the recovery rate of each solution.
In one embodiment, the assay is subjected to a solution stability test, and the specific steps of isocratic elution using a mobile phase are as follows: preparing a test solution containing about 1mg of glimepiride intermediate in each 1ml, injecting samples 0, 12, 24, 36h and 48h after preparation, calculating the contents of a main peak and each impurity peak, and counting the number of impurities.
The analysis and separation method can be realized according to the following method:
(1) chromatographic conditions are as follows: using pentafluorophenyl side chain group bonded silica gel as a stationary phase, the length of a chromatographic column is 150mm, the particle size is 2.7 mu m, a mixed solvent of ammonium dihydrogen phosphate buffer solution and acetonitrile is used as a mobile phase, the flow rate is 0.5ml/min, and the detection wavelength is 225 nm;
(2) preparing a sample solution: diluting a sample to be detected and an impurity reference substance by using a mobile phase and preparing into a sample solution;
(3) separation and analysis: and injecting 20 mu l of sample solution into a high performance liquid chromatograph to complete the determination of the glimepiride intermediate and related substances of impurities thereof.
In the invention, related substances in the glimepiride intermediate 4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-formamido) ethyl ] benzenesulfonamide can be effectively analyzed and monitored through the chromatographic conditions. The analytical method of the invention, such as chromatographic column, detection wavelength, mobile phase proportion setting, has great influence on the separation degree of the benzene sulfonamide and each impurity thereof, each impurity and each substance response signal. Therefore, the inventor comprehensively considers the comprehensive influence of the chromatographic column, the detection wavelength, the type of the mobile phase, the proportion of the mobile phase, the sample diluent and the sample amount on the separation detection, optimizes the detection result, has the advantages of rapid, simple, convenient, accurate and efficient elution, separation and quantitative detection of each impurity in the benzenesulfonamide, can realize the complete separation between the main peak of the benzenesulfonamide and each impurity peak thereof, and the separation degree meets the requirements. The analysis method is convenient to operate, has high sensitivity and stability, and is suitable for separating, detecting and monitoring related substances in the glimepiride intermediate 4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-formamido) ethyl ] benzenesulfonamide, so that the quality of the bulk drug can be effectively controlled. The detection method has the advantages of strong specificity, high sensitivity, convenient operation and effective control of the quality of the medicine.
The mobile phase and the elution time can quickly and efficiently separate the glimepiride intermediate and impurities thereof under the same chromatographic condition, effectively detect the quality of the intermediate and further ensure the quality of the bulk drugs. The analysis method is convenient to operate, fast and effective, and has high sensitivity and stability, so that the basis of research and development and quality detection is provided for the compounds. The analysis method is convenient to operate, short in running time, strong in specificity, high in sensitivity, strong in stability, stable and reliable, has a good linear curve in a low concentration range, has strong repeatability and accuracy, and is not influenced by personnel and instruments.
The method comprehensively considers the comprehensive influence of the type and specification of the chromatographic column, the detection wavelength, the type of the mobile phase, the proportion of the mobile phase, the running time, the sample diluent and the sample amount on the separation detection, optimizes the detection result, has the advantages of fast, simple, convenient, accurate and efficient elution, separation and quantitative detection of each impurity in the glimepiride intermediate, and can realize the complete separation of the impurity peak and the main peak as well as the impurity and the impurity peak. The analysis method is convenient to operate, short in running time, strong in specificity, high in sensitivity, strong in stability, stable and reliable, has a good linear curve in a low concentration range, has strong repeatability and accuracy, and is not influenced by personnel and instruments. The compound provides the basis for research and development and quality detection.
Drawings
FIG. 1 is a system suitability solution.
FIG. 2 shows the test mixed solution.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 comparison of different chromatography columns
High performance liquid chromatography conditions:
agilent Poroshell 120PFP column (150 x 4.6mm,2.7 μm), Welch Ultimate AQ-C18 column (250 x 4.6mm,5 μm), Welch Ultimate XB-C18 column (250 x 4.6mm,5 μm) were each subjected to an assay, using a mobile phase of 0.01mol/L ammonium dihydrogen phosphate buffer solution adjusted to pH 3.5 with phosphoric acid and methanol in a volume ratio of 50:50, with a detection wavelength of 225nm, a flow rate of 1.0ml/min or 0.5ml/min, a column temperature of 25 ℃ and a sample volume of 20 μ L, and isocratic elution was carried out.
Sample preparation:
system applicability solution: a benzenesulfonamide reference substance and an appropriate amount of each impurity reference substance are weighed to prepare a mixed solution containing about 0.1mg of benzenesulfonamide reference substance, 0.5 mu g of impurity A, 0.5 mu g of impurity B, 0.5 mu g of impurity C and 0.5 mu g of impurity D in each 1 ml.
And (3) test operation: and (5) respectively injecting 20 mu l of system applicability solution and recording the chromatogram. The results are shown in Table 1.
TABLE 1 summary of chromatographic column screening results
As can be seen from Table 1, the Agilent Poroshell 120PFP column (150 x 4.6mm,2.7 μm) used in the present invention has better resolution, high component response, high sensitivity, short running time and slow flow rate compared to other columns.
Example 2 comparison of organic phases
High performance liquid chromatography conditions:
methanol as an organic phase and acetonitrile as an organic phase were tested separately on an Agilent Poroshell 120PFP column (150 x 4.6mm,2.7 μm) using a mobile phase of 0.01mol/L ammonium dihydrogen phosphate buffer solution adjusted to pH 3.5 with phosphoric acid and an organic phase at a volume ratio of 50:50, a detection wavelength of 225nm, a flow rate of 0.5ml/min, a column temperature of 25 ℃ and a sample introduction amount of 20 μ L, and isocratic elution was carried out.
The sample formulation was the same as in example 1.
And (3) test operation: and (5) respectively injecting 20 mu l of system applicability solution and recording the chromatogram. The results are shown in Table 2.
TABLE 2 summary of organic phase screening results
As can be seen from Table 2, peaks can be obtained with either methanol or acetonitrile, and the peak time of each component is earlier when acetonitrile which is preferred according to the present invention is used as the organic phase.
Example 3 comparison of mobile phase proportions
High performance liquid chromatography conditions:
the flow phase ratio was tested separately using Agilent Poroshell 120PFP column (150X 4.6mm,2.7 μm), mixed solvent of ammonium dihydrogen phosphate buffer solution (0.01 mol/L) adjusted to pH 3.5 with phosphoric acid and acetonitrile as the mobile phase, detection wavelength 225nm, flow rate 0.5ml/min, column temperature 25 deg.C, sample introduction 20 μ L, and isocratic elution.
The sample formulation was the same as in example 1.
And (3) test operation: and (5) respectively injecting 20 mu l of system applicability solution and recording the chromatogram. The results are shown in Table 3.
Table 3 summary of mobile phase ratio screening results
As can be seen from Table 2, when the ammonium dihydrogen phosphate buffer solution of the present invention is used in a ratio of 0.01mol/L to acetonitrile in a volume ratio of 60: 40 as the mobile phase, the optimum peak time can be achieved and the separability is good. And the process run time is preferably 30 min.
Example 4
High performance liquid chromatography conditions:
the column was an Agilent Poroshell 120PFP column (150 x 4.6mm,2.7 μm) with a volume ratio of 0.01mol/L ammonium dihydrogen phosphate buffer solution adjusted to pH 3.5 with phosphoric acid to acetonitrile of 60: 40 is mobile phase, the detection wavelength is 225nm, the flow rate is 0.5ml/min, the column temperature is 25 ℃, the sample injection amount is 20 mul, and isocratic elution is carried out.
Sample preparation:
glimepiride intermediate control solution: weighing the reference substance, and fixing the volume to the scale by using the mobile phase to obtain a reference solution containing 0.1mg of the benzenesulfonamide in each 1 ml.
Glimepiride intermediate test solutions: the test sample is weighed and the volume is fixed to the scale by using the mobile phase, and the test solution containing 0.1mg of the benzene sulfonamide in each 1ml is obtained.
Component positioning solution: appropriate amounts of the respective component controls were weighed to prepare a localization solution containing about 0.5. mu.g of the impurity A per 1ml, a localization solution containing about 0.5. mu.g of the impurity B per 1ml, a localization solution containing about 0.5. mu.g of the impurity C per 1ml, a localization solution containing about 0.5. mu.g of the impurity D per 1ml, and a localization solution containing about 0.5. mu.g of the benzenesulfonamide per 1ml, respectively.
System applicability solution: a benzenesulfonamide reference substance and an appropriate amount of each impurity reference substance are weighed to prepare a mixed solution containing about 0.1mg of benzenesulfonamide reference substance, 0.5 mu g of impurity A, 0.5 mu g of impurity B, 0.5 mu g of impurity C and 0.5 mu g of impurity D in each 1 ml.
Test mixed solution: a benzene sulfonamide test sample and a proper amount of each impurity reference substance are weighed to prepare a mixed solution containing 0.1mg of the benzene sulfonamide test sample, 0.5 mu g of the impurity A, 0.5 mu g of the impurity B, 0.5 mu g of the impurity C and 0.5 mu g of the impurity D in 1 ml.
And (3) test operation: and respectively sampling 20 mul of each component positioning solution, the system applicability solution and the mixed solution to be tested, and recording the chromatogram for 30 min.
Typical chromatograms are shown in FIG. 1 for system applicability solution chromatograms and FIG. 2 for mixed solution chromatograms.
The results of the invention, which were confirmed for specificity by the system applicability solution, the test mixed solution, and each impurity localization solution, are shown in tables 4 and 5 and fig. 1 and 2.
TABLE 4 results of the specificity test 1
TABLE 5 results of the specificity test 2
As can be seen from Table 3 and FIGS. 1 and 2, under the chromatographic conditions of the present invention, benzenesulfonamide and impurities can be effectively detected, and the retention time (t) of the peaks of benzenesulfonamide and impurities in the chromatogram of the system applicability solution and the chromatogram of the mixed solution to be testedR) Corresponding to the retention time (t) of the main peak in the chromatogram of the corresponding positioning solutionR) And (5) the consistency is achieved.
As can be seen from Table 2 and FIG. 1, the appearance of the peaks in the spectrum of the systematic solution was, in order, impurity A, benzenesulfonamide, impurity B, impurity C and impurity D. The separation degree between the main peak benzene sulfonamide and the impurity B peak is more than 1.5, and the separation degree between the impurity and the impurity is more than 1.5. And the RSD of the 5-needle system applicability solution is less than or equal to 1.0 percent.
Example 5
The HPLC conditions were the same as in example 4.
Sample preparation: and detecting by using a blank mobile phase as a blank solvent.
And (3) test operation: sampling 20 μ l of blank solvent, and recording chromatogram for 30 min.
The invention is researched by using a blank solvent selected by the inventor, and no significant interference peak exists near the peak positions of main peaks, namely benzenesulfonamide, impurity A, impurity B, impurity C and impurity D in a mobile phase chromatogram. The blank solvent proved to be non-interfering with the present invention.
Example 6
The HPLC conditions were the same as in example 4.
Sample preparation:
detection limiting solution: the signal-to-noise ratio of benzenesulfonamide and impurities is about 3: 1 in water.
Quantitative limiting solution: the signal-to-noise ratio of the benzenesulfonamide and various impurities is about 10: 1 in water.
And (3) test operation: and respectively injecting 20 mu l of detection limit solution and 20 mu l of quantification limit solution, and recording the chromatogram for 30 min.
The inventor verifies the detection limit and the quantitative limit of the benzene sulfonamide and each impurity, and the result is shown in table 6.
TABLE 6 test results of detection limit and quantitative limit
As can be seen from Table 6, the detection sensitivity of the main peak benzenesulfonamide of the present invention is high.
Example 7
The HPLC conditions were the same as in example 4.
Sample preparation:
solution one: respectively preparing the benzene sulfonamide and a solution with the limit concentration of each impurity of 120 percent.
Solution II: respectively preparing solution of benzene sulfonamide and each impurity with the limit concentration of 100%.
Solution three: respectively preparing benzene sulfonamide and solution with the limit concentration of each impurity of 60 percent.
Solution four: respectively preparing the benzene sulfonamide and a solution with the limit concentration of each impurity of 20 percent.
Solution five: preparing solution of benzene sulfonamide and each impurity with definite concentration.
And (3) test operation: and respectively taking 20 mu l of each of five linear solutions of the benzenesulfonamide and each impurity, injecting samples respectively, and recording the chromatogram for 30 min.
The inventors verified the linearity and range of benzenesulfonamide and impurities, and the results are shown in table 7.
TABLE 7 results of the linearity and Range test
| Peak name | Linear equation of equations | Coefficient of correlation R2 | Concentration Range (ug/ml) |
| Benzenesulfonamides | y＝0.2683x+0.0157 | 0.9999 | 0.0083-6.024 |
| Impurity A | y＝0.2389x+0.0195 | 0.9996 | 0.0050-6.013 |
| Impurity B | y＝0.1976x+0.0318 | 0.9998 | 0.0106-5.987 |
| Impurity C | y＝0.2269x+0.0177 | 0.9998 | 0.0119-6.045 |
| Impurity D | y＝0.1753x+0.0479 | 0.9996 | 0.0294-5.989 |
As can be seen from Table 7, the linear relationship between the quantitative limit concentration of the benzenesulfonamide and its impurities in the invention and 120% of the limit concentration is good.
Example 8
The HPLC conditions were the same as in example 4.
Sample preparation:
benzenesulfonamide test solutions: the test sample is weighed and the volume is fixed to the scale by using the mobile phase, and the test solution containing about 0.1mg of triphenyl phosphonium bromide in each 1ml is obtained. The repeatability is that 6 parts of test solution is prepared for one experimenter; the intermediate precision was 12 parts of each of 6 parts of test solutions prepared by two laboratory workers.
And (3) test operation: and respectively injecting 20 mu l of test solution, and recording the chromatogram for 30 min.
The inventors verified the repeatability and intermediate precision of triphenylphosphine bromide and each impurity, and the results are shown in Table 8.
TABLE 8 repeatability and intermediate precision test results
From Table 8, the RSD values for the main peak and each impurity repeatability and intermediate precision were found to meet the acceptance criteria. It can be seen that the invention has good repeatability and intermediate precision.
Example 9
The HPLC conditions were the same as in example 4.
Sample preparation:
sample preparation: weighing a benzene sulfonamide test sample and a proper amount of each impurity reference substance, and preparing a sample adding and recovering solution with the limit concentration of 20%, a sample adding and recovering solution with the limit concentration of 50% and a sample adding and recovering solution with the limit concentration of 120%.
And (3) test operation: and respectively injecting 20 μ l of each of the 20% sample-adding recovery solution, the 50% sample-adding recovery solution and the 120% sample-adding recovery solution, and recording the chromatogram for 30 min.
The inventors verified the accuracy of each impurity of benzenesulfonamide, and the results are shown in table 9.
TABLE 9 accuracy test results
From table 9, it can be seen that the normalized recovery rate of each impurity meets the acceptable standard, indicating that the invention can accurately and stably detect the content of each impurity.
Example 10
The HPLC conditions were the same as in example 4.
Sample preparation:
benzenesulfonamide test solutions: the test sample is weighed and the volume is fixed to the scale by using the mobile phase, and the test solution containing about 0.1mg of triphenyl phosphonium bromide in each 1ml is obtained.
And (3) test operation: sampling 20 μ l of test solution at 0, 12, 24, 36, and 48h, respectively, and recording chromatogram for 30 min.
The inventors of the present invention verified the solution stability of benzenesulfonamide and each impurity.
The result shows that the sample solution is stable within 48 hours without detecting more impurities after being placed for 48 hours.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. An analytical method for determining related substances in a glimepiride intermediate by utilizing HPLC, which is characterized by comprising the following steps:
(1) chromatographic conditions are as follows: taking an octadecylsilane chemically bonded silica chromatographic column as a stationary phase, and taking a phosphate buffer solution and an organic phase in a volume ratio of (50-70): (30-50) the formed mixed solvent is a mobile phase; the flow rate is 0.4-1.0 ml/min, the column temperature is 20-30 ℃, and the detection wavelength is 225 nm.
2. The analytical method of claim 1, wherein: and diluting the sample to be detected with a mobile phase to prepare a sample solution, preferably, 0.05-0.2 mg/ml of solution.
3. The analytical method of claim 1, wherein: the glimepiride intermediate is 4- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-formamido) ethyl ] benzenesulfonamide, and the related substances are as follows:
impurity A: 3-ethyl-4-methyl-3-pyrrolin-2-one;
impurity B: 3- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) -ethyl ] -benzenesulfonamide;
impurity C: 2- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) -ethyl ] -benzenesulfonamide;
impurity D: 3-ethyl-4-methyl-2-oxo-3-pyrroline-N- (2-phenylethyl) carboxamide.
4. An assay method according to claim 1, wherein the length of the chromatographic column is 150 to 250mm, preferably 150 mm; the diameter is 4-5 mm, and the preferred diameter is 4.6 mm; the particle size of the filler is 2.7-5.0 μm, and the preferred particle size is 2.7 μm; preferably, the chromatographic column is an Agilent Poroshell 120PFP pentafluorophenyl column.
5. The method according to claim 1, wherein the phosphate buffer solution is a ammonium dihydrogen phosphate buffer solution, preferably, the concentration of the ammonium dihydrogen phosphate buffer solution is 0.01 to 0.1mol/L, and preferably, the pH of the ammonium dihydrogen phosphate buffer solution is 3.0 to 5.0.
6. The analytical method according to claim 1, characterized in that the organic phase is methanol or acetonitrile, preferably acetonitrile.
7. The analytical method according to claim 1, wherein the volume ratio of the mobile phase phosphate buffer solution to the acetonitrile is 60: 40; preferably, the elution time is 25-60 min, more preferably 30 min.
8. The analytical method of claim 1, wherein the flow rate of the mobile phase is 0.5 ml/min.
9. The analytical method according to claim 1, wherein the column temperature is 25 ℃.
10. The method according to claim 1, wherein the amount of the sample is 10 to 20. mu.l, preferably 20. mu.l.
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