Method for detecting 6-chloro-1, 3-indoline-2-ketone related substances
阅读说明：本技术 一种用于检测6-氯-1,3-二氢吲哚-2-酮有关物质的方法 (Method for detecting 6-chloro-1, 3-indoline-2-ketone related substances ) 是由 陈年根 于 2021-07-29 设计创作，主要内容包括：本发明提供一种用于检测6-氯-1,3-二氢吲哚-2-酮有关物质的方法,采用的色谱条件为：色谱柱为InertsilODS-3(4.6×250mm,5μm),柱温为25～35℃,检测波长210～220nm,流动相为以磷酸盐缓冲液体(磷酸调节pH＝2.9～3.1)为A相,以乙腈为B相,流速为0.9～1.1ml/min。利用本发明的方法可以准确地进行6-氯-1,3-二氢吲哚-2-酮中有关物质定量分析,可以有效将6-氯-1,3-二氢吲哚-2-酮与杂质分离,专属性、精密度、耐用性均符合要求,检测效果良好。(The invention provides a method for detecting 6-chloro-1, 3-indoline-2-ketone related substances, which adopts chromatographic conditions as follows: the chromatographic column is InertsilODS-3(4.6 multiplied by 250mm, 5 mu m), the column temperature is 25-35 ℃, the detection wavelength is 210-220 nm, the mobile phase is phosphate buffer liquid (the pH value is adjusted to be 2.9-3.1) as the phase A, the acetonitrile as the phase B, and the flow rate is 0.9-1.1 ml/min. The method can accurately perform quantitative analysis on related substances in the 6-chloro-1, 3-indoline-2-ketone, can effectively separate the 6-chloro-1, 3-indoline-2-ketone from impurities, meets the requirements on specificity, precision and durability, and has good detection effect.)
1. A method for detecting 6-chloro-1, 3-indolin-2-one related substances, which is characterized by comprising the following steps:
s1, preparing a test solution:
taking a 6-chloro-1, 3-indoline-2-ketone sample, dissolving the sample by using a solvent and preparing a test solution;
s2, preparing an impurity reference stock solution:
dissolving impurity A, impurity B, impurity C, impurity D and impurity E standard reference substances with a solvent to prepare an impurity reference substance stock solution;
s3, preparing a mixed solution:
taking a 6-chloro-1, 3-indoline-2-ketone sample, adding an impurity A, an impurity B, an impurity C, an impurity D and an impurity E reference substance stock solution, and adding a solvent to prepare a mixed solution;
s4, respectively sucking the equal amount of sample solution and mixed solution, and injecting into a high performance chromatograph for determination; the determination conditions of the high performance liquid chromatography comprise: the chromatographic column is Inertsil ODS-3, the column temperature is 25-35 ℃, the detection wavelength is 210-220 nm, the mobile phase takes phosphate buffer liquid as phase A, and acetonitrile as phase B; the mobile phase was eluted using the following procedure:
。
2. The method for detecting 6-chloro-1, 3-indolin-2-one related substance according to claim 1, wherein the solvent is acetonitrile.
3. The method for detecting 6-chloro-1, 3-indolin-2-one related substance according to claim 2, wherein in step S1, the mass-to-volume ratio g/l of 6-chloro-1, 3-indolin-2-one and acetonitrile in the test solution is 1: 1.
4. the method for detecting 6-chloro-1, 3-indolin-2-one related substance according to claim 1, wherein the structural formula of the impurity a is shown as (a); the structural formula of the impurity B is shown as (B); the structural formula of the impurity C is shown as (C); the structural formula of the impurity D is shown as (D); the structural formula of the impurity E is shown as (E);
5. the method for detecting 6-chloro-1, 3-indolin-2-one related substance according to claim 2, wherein in step S2, the mass volume ratios g/l of impurity a, impurity B, impurity C, impurity D, impurity E and acetonitrile in the impurity control stock solution are all 1: 1.
6. the method for detecting 6-chloro-1, 3-indolin-2-one related substances according to claim 2, wherein in step S3, the mass-to-volume ratio g/l of 6-chloro-1, 3-indolin-2-one and acetonitrile in the mixed solution is 0.01 to 2.0: 1; the mass volume ratio g/l of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the acetonitrile in the mixed solution is 0.01: 1.
7. the method for detecting 6-chloro-1, 3-indolin-2-one related substance as set forth in claim 1, wherein the Inertsil ODS-3 has a length of 250mm, a diameter of 4.6mm, and a filler particle size of 5 μm.
8. The method for detecting 6-chloro-1, 3-indolin-2-one related substance according to claim 1, wherein the flow rate of the mobile phase in step S4 is 0.9-1.1 ml/min.
9. The method for detecting 6-chloro-1, 3-indolin-2-one related substance according to claim 1 or 8, wherein in step S4, the column temperature is 30 ℃, the detection wavelength is 215nm, and the flow rate of the mobile phase is 1.0 ml/min.
10. The method for detecting 6-chloro-1, 3-indolin-2-one related substance according to claim 1, wherein the pH of the mobile phase A is 2.9 to 3.1.
Technical Field
The invention relates to a high performance liquid chromatography analysis method, in particular to a method for detecting related substances of 6-chloro-1, 3-indoline-2-ketone.
Background
Ziprasidone (Ziprasidone) was developed by feverer in 1987, and oral and intramuscular dosage forms were marketed in sweden in 1998 and 9 months 2000, respectively. Ziprasidone hydrochloride capsules from this company were approved for sale in the united states on day 2/25 of 2001; ziprasidone mesylate injection was approved for sale in the united states under the trade name Geodon, 21/6/2002, and thereafter, was marketed in over 89 countries, sweden, new zealand, etc., in succession. Indications for ziprasidone include schizophrenia, acute manic episodes of bipolar affective disorder and mixed episodes.
In 2004, clinical data showed that Geodon for treating schizophrenia had unique advantages in the aspects of effectiveness, dose flexibility, adverse reaction tolerance and the like compared with the original drug, and the sales of the drug was strongly increased. In 2006, this drug became the fastest growing atypical antipsychotic in the united states. In 2009, Geodon listed us brand drug retail sales TOP200, with sales of $ 8.74 billion. In 2011, Geodon has a sales volume of about $ 8.59 billion in the united states.
The 6-chloro-1, 3-indoline-2-ketone (1) is an important intermediate in the synthesis step of ziprasidone and is mainly prepared by condensation, decarboxylation, reduction and cyclization of 1, 4-dichloro-2-nitrobenzene.
In the prior art, no corresponding analysis and detection method is available for impurities in 6-chloro-1, 3-indoline-2-ketone, and the existing impurities can be conducted to ziprasidone hydrochloride to influence the quality of the product, so that an intermediate related substance detection method which is simple and convenient to operate, high in sensitivity and good in reproducibility is urgently needed to be established for continuously improving the safety and effectiveness of the medicine.
According to the synthetic route of 6-chloro-1, 3-indoline-2-ketone, the chemical structural formulas of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E which are possibly existed are respectively as follows:
disclosure of Invention
In view of the above, the present invention aims to provide a method for analyzing and detecting 6-chloro-1, 3-indolin-2-one and related impurities, which is used for monitoring quality. The method has the advantages of high separation degree, simple operation, short analysis time, good repeatability and durability, and stable and reliable result.
In order to achieve the purpose of the invention, the inventor conducts a great deal of verification, and the technical scheme of the invention is realized as follows:
a method for detecting 6-chloro-1, 3-indolin-2-one related substances, which adopts high performance liquid chromatography to carry out quantitative detection on 6-chloro-1, 3-indolin-2-one and related substances, wherein the conditions of the high performance liquid chromatography comprise: the chromatographic column is Inertsil ODS-3(4.6 multiplied by 250mm, 5 mu m), the mobile phase A is a phosphate buffer solution system (pH is 2.9-3.1), the mobile phase B is acetonitrile, the detection wavelength is 210-220 nm, the column temperature is 25-35 ℃, and the flow rate is 0.9-1.1 ml/min.
Preferably, the phosphate buffer solution system is a potassium dihydrogen phosphate solution prepared by mixing potassium dihydrogen phosphate and water and adjusting the pH value to 2.9-3.1 by using phosphoric acid.
Preferably, the pH of the potassium dihydrogen phosphate solution is 3.0.
Preferably, the potassium dihydrogen phosphate solution is a 0.01mol/L potassium dihydrogen phosphate solution.
Preferably, the detection wavelength is 215 nm.
Preferably, the column temperature is 30 ℃.
Preferably, the flow rate is 1.0 ml/min.
Preferably, the method for detecting 6-chloro-1, 3-indolin-2-one related substances according to the invention can be realized by the following steps:
s1, preparing a test solution:
taking a proper amount of a 6-chloro-1, 3-indoline-2-ketone sample, dissolving the sample by using a solvent and preparing a test solution;
s2, preparing an impurity reference stock solution:
dissolving impurity A, impurity B, impurity C, impurity D and impurity E standard reference substances with a solvent to prepare an impurity reference substance stock solution;
s3, preparing a mixed solution:
taking a 6-chloro-1, 3-indoline-2-ketone sample, adding an impurity A, an impurity B, an impurity C, an impurity D and an impurity E reference substance stock solution, and adding a solvent to prepare a mixed solution;
s4, setting conditions of a high performance liquid chromatograph, respectively sucking equal amounts of a test solution and a mixed solution, and injecting the test solution and the mixed solution into the high performance liquid chromatograph for determination;
wherein the conditions of the high performance liquid chromatograph are as follows
A chromatographic column: inertsil ODS-3 (4.6X 250mm, 5 μm);
phosphate buffer (0.01 mol/L potassium dihydrogen phosphate solution is taken, and the pH value is adjusted to 3.0 by phosphoric acid) is used as a mobile phase A, and acetonitrile is used as a mobile phase B.
Detection wavelength: 210-220 nm;
column temperature: 25-35 ℃;
flow rate: 0.9-1.1 ml/min;
elution procedure:
time (min)
Mobile phase A (%)
Mobile phase B (%)
0
60
40
10
30
70
25
30
70
30
60
40
60
60
40
Preferably, the solvent is acetonitrile.
Preferably, the test solution contains 1mg of 6-chloro-1, 3-indolin-2-one per 1ml of the test solution.
Preferably, the impurity control stock solution contains 1mg each of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E per 1mL of the impurity control stock solution.
Preferably, each 1ml of the mixed solution contains 0.01-2 mg of 6-chloro-1, 3-indoline-2-ketone, and 10 mug of each of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E.
Preferably, 1mg of 6-chloro-1, 3-indolin-2-one is contained per 1ml of the mixed solution, and 10. mu.g of each of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E is contained.
Preferably, the structural formula of the impurity A is shown as (a); the structural formula of the impurity B is shown as (B); the structural formula of the impurity C is shown as (C); the structural formula of the impurity D is shown as (D); the structural formula of the impurity E is shown as (E);
preferably, the same amount of the sample solution and the mixed solution is aspirated as 10. mu.L of the sample solution and the mixed solution, respectively.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention provides a method for measuring 6-chloro-1, 3-indoline-2-ketone related substances, a method for separating and detecting 6-chloro-1, 3-indoline-2-ketone, impurity A, impurity B, impurity C, impurity D and impurity E, which adopts chromatographic conditions different from the prior art, a certain distance exists between 6-chloro-1, 3-indoline-2-ketone and impurity in HPLC (high performance liquid chromatography) map, 6-chloro-1, 3-indoline-2-ketone related substances can be accurately measured, 6-chloro-1, 3-indoline-2-ketone can be effectively separated from impurity A, impurity B, impurity C, impurity D and impurity E, the separation degree is higher than 1.5, in destructive tests, 6-chloro-1, 3-indolin-2-one and impurities are also ensured to be free from interference. The method has the advantages of simple operation, good repeatability and durability, and stable and reliable result, and can be used for quality control of 6-chloro-1, 3-indoline-2-ketone, and provide effective guarantee for the quality of the final product.
(2) The separation detection method disclosed by the invention has the advantages that the aspects of separation degree, specificity, precision, durability and the like are verified in detail, all verification results meet the requirements of relevant regulations and guiding principles, and the actual detection effect is good.
(3) In the high performance liquid chromatography according to the present invention, the effective values are within the range included in the measurement conditions, that is, the following values: after any value is taken within each parameter range, the 6-chloro-1, 3-indoline-2-ketone related substance can be accurately detected, and the impurities A, B, C, D and E can be effectively separated. In the actual detection process, the method is convenient for detection personnel to adjust the parameters and avoid the influence of human errors on the detection result, and the method has strong practicability, simple and quick detection process and is suitable for popularization and application.
Drawings
FIG. 1 is an HPLC chromatogram of a blank solution of example 1 of the present invention; wherein the blank solution is 10 mu L of acetonitrile without a sample;
FIG. 2 is an HPLC chromatogram of a mixed solution of example 1 of the present invention;
FIG. 3 is an HPLC chromatogram of a test solution of example 1 of the present invention;
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
The invention provides a method for detecting 6-chloro-1, 3-indoline-2-ketone related substances, wherein impurities comprise an impurity A, an impurity B, an impurity C, an impurity D and an impurity E, and the impurities are mainly used for separating and detecting the impurity A, the impurity B, the impurity C, the impurity D and the impurity E in the 6-chloro-1, 3-indoline-2-ketone.
In each of the following 5 exemplary examples, a sample of 6-chloro-1, 3-indolin-2-one was taken from the same lot (190923 lots) and used for methodological studies.
Example 1 System suitability test
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: inertsil ODS-3 (4.6X 250mm, 5 μm);
phosphate buffer (0.01 mol/L potassium dihydrogen phosphate solution is taken, and the pH value is adjusted to 3.0 by phosphoric acid) is used as a mobile phase A, and acetonitrile is used as a mobile phase B.
Detection wavelength: 215 nm;
column temperature: 30 ℃;
flow rate: 1.0 ml/min;
elution procedure:
time (min)
Mobile phase A (%)
Mobile phase B (%)
0
60
40
10
30
70
25
30
70
30
60
40
60
60
40
The experimental steps are as follows:
s1, preparing a test solution:
taking a proper amount of a 6-chloro-1, 3-indoline-2-ketone sample, dissolving the sample with acetonitrile, and preparing a test solution containing 1mg of 6-chloro-1, 3-indoline-2-ketone per 1 mL;
s2, preparing an impurity reference stock solution:
dissolving impurity A, impurity B, impurity C, impurity D and impurity E standard reference substances with acetonitrile, and preparing 1mg impurity reference substance stock solution containing impurity A, impurity B, impurity C, impurity D and impurity E in each 1 mL;
s3, preparing a mixed solution:
taking a 6-chloro-1, 3-indoline-2-ketone sample, adding an impurity A, an impurity B, an impurity C, an impurity D and an impurity E reference substance stock solution, and adding acetonitrile to prepare a mixed solution containing 1mg of 6-chloro-1, 3-indoline-2-ketone and 10 micrograms of each of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E per 1 mL;
s4, setting conditions of a high performance liquid chromatograph, respectively sucking 10 mu L of sample solution and mixed solution, injecting the sample solution and the mixed solution into the high performance liquid chromatograph, and inspecting the separation condition of each impurity and main component;
from the data results in the table above, the retention time of the main component is 23.220min, and the separation degree of each impurity and the main component peak is greater than 1.5, which indicates that the separation degree can meet the analysis requirement under the condition.
Example 2 precision test
Experimental apparatus and conditions:
the instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: inertsil ODS-3 (4.6X 250mm, 5 μm);
phosphate buffer (0.01 mol/L potassium dihydrogen phosphate solution is taken, and the pH value is adjusted to 3.0 by phosphoric acid) is used as a mobile phase A, and acetonitrile is used as a mobile phase B.
Detection wavelength: 215 nm;
column temperature: 30 ℃;
flow rate: 1.0 ml/min;
elution procedure:
time (min)
Mobile phase A (%)
Mobile phase B (%)
0
60
40
10
30
70
25
30
70
30
60
40
60
60
40
The experimental steps are as follows:
s1, preparing a test solution:
taking a proper amount of a 6-chloro-1, 3-indoline-2-ketone sample, dissolving the sample with acetonitrile, and preparing a test solution containing 1mg of 6-chloro-1, 3-indoline-2-ketone per 1 mL;
s2, preparing an impurity reference stock solution:
dissolving impurity A, impurity B, impurity C, impurity D and impurity E standard reference substances with acetonitrile, and preparing 1mg impurity reference substance stock solution containing impurity A, impurity B, impurity C, impurity D and impurity E in each 1 mL;
s3, preparing a mixed solution:
taking a 6-chloro-1, 3-indoline-2-ketone sample, adding an impurity A, an impurity B, an impurity C, an impurity D and an impurity E reference substance stock solution, and adding acetonitrile to prepare a mixed solution containing 10 mu g of each of the 6-chloro-1, 3-indoline-2-ketone, the impurity A, the impurity B, the impurity C, the impurity D and the impurity E in each 1 mL;
s4, setting conditions of a high performance liquid chromatograph, respectively sucking 10 mu L of test sample solution and mixed solution, injecting the test sample solution and the mixed solution into the high performance liquid chromatograph, respectively preparing 6 parts of test sample for each person, measuring related substances according to an external standard method, and calculating the relative standard deviation of the 12 parts of related substances;
as can be seen from the data in the table, 6 parts of the sample are tested repeatedly on different instruments by different persons at different times, the impurity A is detected, other known impurities are not detected, and the results of the related substances of the 6 parts of the sample are consistent; the results of 12 groups of data are basically consistent, and the intermediate precision is good.
EXAMPLE 3 durability test
Experimental apparatus and conditions:
the instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: inertsil ODS-3 or YMC-Pack ODS-A (4.6X 250mm, 5 μm);
taking a phosphate buffer solution (taking 0.01mol/L potassium dihydrogen phosphate solution, adjusting the pH value to 2.9-3.1 by using phosphoric acid) as a mobile phase A, and taking acetonitrile as a mobile phase B;
detection wavelength: 210-220 nm;
column temperature: 25-35 ℃;
flow rate: 0.9-1.1 ml/min;
elution procedure:
time (min)
Mobile phase A (%)
Mobile phase B (%)
0
60
40
10
30
70
25
30
70
30
60
40
60
60
40
The experimental steps are as follows:
s1, preparing a test solution:
taking a proper amount of a 6-chloro-1, 3-indoline-2-ketone sample, dissolving the sample with acetonitrile, and preparing a test solution containing 1mg of 6-chloro-1, 3-indoline-2-ketone per 1 mL;
s2, preparing an impurity reference stock solution:
dissolving impurity A, impurity B, impurity C, impurity D and impurity E standard reference substances with acetonitrile, and preparing 1mg impurity reference substance stock solution containing impurity A, impurity B, impurity C, impurity D and impurity E in each 1 mL;
s3, preparing a mixed solution:
taking a 6-chloro-1, 3-indoline-2-ketone sample, adding an impurity A, an impurity B, an impurity C, an impurity D and an impurity E reference substance stock solution, and adding acetonitrile to prepare a mixed solution containing 10 mu g of each of the 6-chloro-1, 3-indoline-2-ketone, the impurity A, the impurity B, the impurity C, the impurity D and the impurity E in each 1 mL;
s4, setting conditions of a high performance liquid chromatograph, respectively sucking 10 mu L of test sample solution and mixed solution, injecting the test sample solution and the mixed solution into the high performance liquid chromatograph, and calculating related substances of the test sample;
note that1: the chromatographic conditions under normal conditions were the same as in example 1;
note that2: taking normal conditions as a standard, when the flow rate, the column temperature, the detection wavelength, the pH or the chromatographic column in the detection conditions are changed, other conditions are unchanged;
as can be seen from the data in the table above, when the column temperature changes by + -5 ℃, the flow rate changes by + -0.1 ml/min, the detection wavelength changes by + -5 nm, the mobile phase ApH changes by + -0.1, and the chromatographic conditions such as the chromatographic column conditions and the like are slightly changed, the system applicability meets the requirements, the detection results of the related substances have no obvious difference, and the durability is good.
Example 4 specificity test
Experimental apparatus and conditions:
the instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: inertsil ODS-3 (4.6X 250mm, 5 μm);
phosphate buffer (0.01 mol/L potassium dihydrogen phosphate solution is taken, and the pH value is adjusted to 3.0 by phosphoric acid) is used as a mobile phase A, and acetonitrile is used as a mobile phase B.
Detection wavelength: 215 nm;
column temperature: 30 ℃;
flow rate: 1.0 ml/min;
elution procedure:
time (min)
Mobile phase A (%)
Mobile phase B (%)
0
60
40
10
30
70
25
30
70
30
60
40
60
60
40
The experimental steps are as follows:
s1, preparing a test solution:
taking a proper amount of a 6-chloro-1, 3-indoline-2-ketone sample, dissolving the sample with acetonitrile, and preparing a test solution containing 1mg of 6-chloro-1, 3-indoline-2-ketone per 1 mL;
s2, preparing an impurity reference stock solution:
dissolving impurity A, impurity B, impurity C, impurity D and impurity E standard reference substances with acetonitrile, and preparing 1mg impurity reference substance stock solution containing impurity A, impurity B, impurity C, impurity D and impurity E in each 1 mL;
s3, preparing a mixed solution:
taking a 6-chloro-1, 3-indoline-2-ketone sample, adding an impurity A, an impurity B, an impurity C, an impurity D and an impurity E reference substance stock solution, and adding acetonitrile to prepare a mixed solution containing 1mg of 6-chloro-1, 3-indoline-2-ketone and 10 micrograms of each of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E per 1 mL;
s4, dissolving in acetonitrile, destroying under various degradation conditions, and making into 1mg per 1ml solution.
S5, setting conditions of the high performance liquid chromatograph, and respectively sucking 10 mu L of sample solution, mixed solution and product solution to be injected into the high performance liquid chromatograph;
the experimental result shows that the main component has stable property, has no obvious degradation under the conditions and only slightly degrades under the conditions of acid and alkali; other conditions are relatively stable, and the separation degree of the generated degradation impurities, the main peak and each known impurity meets the requirement; under each degradation condition, the purity angle of the main peak of the main component is smaller than a threshold value; the conservation of materials is between 90 and 110 percent before and after degradation. The experimental results show that the method can effectively detect all known impurities and unknown impurities, and the separation degrees among the impurities and between the impurities and the main peak meet the requirements, which indicates that the method has better specificity.
Example 5 detection Limit test
Experimental apparatus and conditions:
the instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: inertsil ODS-3 (4.6X 250mm, 5 μm);
phosphate buffer (0.01 mol/L potassium dihydrogen phosphate solution is taken, and the pH value is adjusted to 3.0 by phosphoric acid) is used as a mobile phase A, and acetonitrile is used as a mobile phase B.
Detection wavelength: 215 nm;
column temperature: 30 ℃;
flow rate: 1.0 ml/min;
elution procedure:
time (min)
Mobile phase A (%)
Mobile phase B (%)
0
60
40
10
30
70
25
30
70
30
60
40
60
60
40
The experimental steps are as follows:
s1, preparing an impurity reference stock solution:
dissolving impurity A, impurity B, impurity C, impurity D and impurity E standard reference substances with acetonitrile, and preparing 1mg impurity reference substance stock solution containing impurity A, impurity B, impurity C, impurity D and impurity E in each 1 mL;
s1, preparing a mixed solution:
taking a 6-chloro-1, 3-indoline-2-ketone sample, adding an impurity A, an impurity B, an impurity C, an impurity D and an impurity E reference substance stock solution, and adding acetonitrile to prepare a mixed solution containing 10 mu g of each of the 6-chloro-1, 3-indoline-2-ketone, the impurity A, the impurity B, the impurity C, the impurity D and the impurity E in each 1 mL;
and S3, setting conditions of a high performance liquid chromatograph, diluting the mixed solution with acetonitrile step by step, precisely measuring 10 mu L of each solution, injecting the solution into the liquid chromatograph, and recording a chromatogram. The signal-to-noise ratio is not less than 3 as the detection limit.
Sample (I)
S/N
Detection limit (ng)
Principal component
3.77
0.49
Impurity A
3.53
0.23
Impurity B
4.04
0.50
Impurity C
3.35
0.53
Impurity D
3.50
0.50
Impurity E
3.27
0.36
As can be seen from the data results, the sensitivity of each impurity can meet the requirements.
In summary, from the detection results of examples 1 to 5, it is known that, in the detection process of the 6-chloro-1, 3-indolin-2-one related substance, the results of the 6-chloro-1, 3-indolin-2-one related substance can be unaffected and the detection results are effective and accurate after the flow rate, the column temperature, the mobile phase pH, the wavelength, the chromatographic column and the like are adjusted within the range of the parameters of the chromatographic conditions of the present invention, which indicates that the separation and detection method of the present invention can accurately detect the 6-chloro-1, 3-indolin-2-one related substance and can effectively separate the 6-chloro-1, 3-indolin-2-one from the impurity a, the impurity B, the impurity C, the impurity D and the impurity E.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.