CN111983056A - Method for separating and measuring related substances of tofacitinib intermediate by using HPLC (high performance liquid chromatography) - Google Patents
Method for separating and measuring related substances of tofacitinib intermediate by using HPLC (high performance liquid chromatography) Download PDFInfo
- Publication number
- CN111983056A CN111983056A CN202010739267.8A CN202010739267A CN111983056A CN 111983056 A CN111983056 A CN 111983056A CN 202010739267 A CN202010739267 A CN 202010739267A CN 111983056 A CN111983056 A CN 111983056A
- Authority
- CN
- China
- Prior art keywords
- related substances
- solution
- tofacitinib intermediate
- mobile phase
- tofacitinib
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a method for separating and measuring related substances of a tofacitinib intermediate by using HPLC (high performance liquid chromatography), which comprises the following steps of: octadecylsilane chemically bonded silica is used as a filling agent, 0.005-0.03 mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is carried out, the flow rate is 0.8-1.2 ml/min, the column temperature is 25-40 ℃, the detection wavelength is 205-230 nm, and an ultraviolet detector is used for detecting related substances of the tofacitinib intermediate. The method comprehensively considers the analysis column, the mobile phase, the gradient elution program, the flow rate and the comprehensive influence of the column temperature on the separation detection, optimizes the detection result, has the advantages of rapidness, simplicity, convenience, high sensitivity, accuracy, reliability and wide applicability, and is suitable for separating and determining the related substances of the tofacitinib intermediate, thereby effectively controlling the quality of the medicine.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for separating and determining related substances of a tofacitinib intermediate by using HPLC (high performance liquid chromatography).
Background
Tofacitinib (trade name Xeljanz), an oral small molecule Janus kinase (JAK) inhibitor developed by seires corporation in the united states, was approved by the Food and Drug Administration (FDA) in 2012 and 11 months for treating adult patients with moderate to severe rheumatoid arthritis who have inadequate or intolerant methotrexate response.
The 4-hydroxy pyrrolopyrimidine is a tofacitinib intermediate, and the molecular formula is as follows: c6H5N3O, molecular weight: 135.12, the chemical structural formula is shown as the following formula (1):
in the preparation process of the intermediate, a plurality of impurities are generated due to various factors such as starting materials, synthesis process, degradation and the like, wherein the impurities A, B, C, D and E are easy to generate in the synthesis process and are mainly considered as impurities in related substance projects, and the limit requirements of the impurities are all not more than 0.50 wt%.
Impurity A is a byproduct, and the molecular formula is as follows: c4H4N3NaOS, molecular weight: 165.15, the chemical formula is shown as the following formula (2):
impurity B is a byproduct, and the molecular formula is as follows: c4H5N3O, molecular weight: 111.1, chemical structural formula as following formula (3):
impurity C is a byproduct, and the molecular formula is as follows: c6H5N3OS, molecular weight: 167.19, the chemical formula is represented by the following formula (4):
impurity D is an intermediate, molecular formula: c10H17N3O3Molecular weight: 227.26, the chemical formula is shown as the following formula (5):
impurity E is an intermediate, molecular formula: c10H17N3O3S, molecular weight: 259.33, the chemical formula is shown as the following formula (6):
the analysis and detection of the intermediate plays an important role in reaction control and yield improvement, and simultaneously directly influences the quality of a final product, so that the establishment of a stable and effective analysis and detection method with simple operation is very necessary for the analysis and detection of the tofacitinib intermediate. In the prior art, no analysis method suitable for quickly, simply and accurately analyzing and detecting related substances of tofacitinib intermediate exists. Therefore, there is a need for further improvement and optimization of the assay method for tofacitinib intermediate related substances.
Disclosure of Invention
In order to overcome the technical problems in the prior art, the inventor of the present invention has conducted a great deal of intensive research, and thus provides a method for separating and determining related substances of tofacitinib intermediate by using HPLC, which has the advantages of being fast, simple, convenient, high in sensitivity, accurate and reliable.
The technical scheme adopted by the invention is as follows:
a method for separating and determining related substances of a tofacitinib intermediate by using HPLC (high performance liquid chromatography), which comprises the following steps: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, using 0.005-0.03 mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile, methanol or acetonitrile-methanol as a mobile phase B, wherein the flow rate is 0.8-1.2 ml/min, the column temperature is 25-40 ℃, and the volume ratio of the acetonitrile to the methanol is 40: 60-60: 40; and detecting related substances of the tofacitinib intermediate by adopting an ultraviolet detector, wherein the detection wavelength of the ultraviolet detector is 205-230 nm.
The invention relates to a method for separating and determining tofacitinib intermediate related substances by using HPLC, wherein the gradient elution condition is as follows:
| time in minutes | Mobile phase A, volume% | Mobile phase B, volume% |
| 0 | 90～97 | 3～10 |
| 5 | 90～97 | 3～10 |
| 55 | 25～35 | 65～75 |
| 56 | 90～97 | 3～10 |
| 70 | 90～97 | 3～10 |
Therefore, separation among impurities and between the impurities and the main peak can be effectively realized, and the separation degree is good. Impurity D and an unknown impurity in the sample are difficult to separate, and the separation degree is not good by adopting other data.
The invention relates to a method for separating and determining tofacitinib intermediate related substances by using HPLC, wherein the gradient elution condition is as follows:
| time in minutes | Mobile phase A, volume% | Mobile phase B, volume% |
| 0 | 95 | 5 |
| 5 | 95 | 5 |
| 55 | 30 | 70 |
| 56 | 95 | 5 |
| 70 | 95 | 5 |
The separation obtained is the best and the peak shape is the best.
The invention relates to a method for separating and measuring related substances of a tofacitinib intermediate by using HPLC (high performance liquid chromatography), wherein a mobile phase A is a potassium dihydrogen phosphate solution with the concentration of 0.01mol/L, and a mobile phase B is acetonitrile. Thus, the degree of separation and the number of theoretical plates are both good.
The invention relates to a method for separating and measuring tofacitinib intermediate related substances by using HPLC, wherein the detection wavelength of an ultraviolet detector is 210 nm. This can further improve the detection sensitivity of impurities.
The invention relates to a method for separating and measuring tofacitinib intermediate related substances by using HPLC (high performance liquid chromatography), wherein the tofacitinib intermediate related substances comprise one or more of impurities A, B, C, D and E, and the specific structural formula is as follows:
the invention relates to a method for separating and measuring related substances of a tofacitinib intermediate by using HPLC (high performance liquid chromatography), wherein the length of a chromatographic column is 150-250 mm; the particle size of the filler is 1.8-5 μm.
The invention relates to a method for separating and measuring tofacitinib intermediate related substances by using HPLC, wherein the length of a chromatographic column is 250 mm. This can further improve the degree of separation.
The invention relates to a method for separating and measuring tofacitinib intermediate related substances by using HPLC, wherein the particle size of a filling agent is 5 mu m. This can further improve the degree of separation.
The method for separating and measuring related substances of the tofacitinib intermediate by using HPLC (high performance liquid chromatography) is characterized in that the flow rate is 1.0ml/min, and the column temperature is 30 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the method for separating and determining the related substances of the tofacitinib intermediate by using HPLC comprehensively considers the comprehensive influence of an analytical column, a mobile phase, a gradient elution program, flow rate and column temperature on separation and detection, so that the detection result is optimized, the impurity A, the impurity B, the impurity C, the impurity D and the impurity E in the tofacitinib intermediate can be quickly and efficiently separated under the same chromatographic condition, and the detection method has the advantages of high sensitivity, strong specificity, strong accuracy, quickness, simplicity and convenience in operation, can effectively control the quality of a medicine, and is suitable for separating and determining the related substances of the tofacitinib intermediate. The mobile phase A is 0.005-0.03 mol/L potassium dihydrogen phosphate solution, and the concentration of the potassium dihydrogen phosphate solution is too low, so that the separation degree of impurities A, B is not good, and the chromatographic column can be damaged if the concentration is too high.
Drawings
FIG. 1 is a chromatogram of a blank solution tested under the conditions of example 1 in accordance with the present invention;
FIG. 2 is a chromatogram of a system suitability solution tested under the conditions of example 1 in the present invention;
FIG. 3 is a chromatogram of a test solution tested under the conditions of example 1 in accordance with the present invention;
FIG. 4 is a chromatogram of a limiting quantitation solution detected under the conditions of example 1 in the present invention;
FIG. 5 is a chromatogram of a quantitation limit + detection limit solution detected under the conditions of example 1 in the present invention;
FIG. 6 is a chromatogram of a detection limiting solution detected under the conditions of example 1 in the present invention;
FIG. 7 is a chromatogram of a blank solution tested under the conditions of example 2 in accordance with the present invention;
FIG. 8 is a chromatogram of a system suitability solution tested under the conditions of example 2 in the present invention;
FIG. 9 is a chromatogram of a test solution tested under the conditions of example 2 in accordance with the present invention;
FIG. 10 is a chromatogram of a limiting quantitation solution detected under the conditions of example 2 in the present invention;
FIG. 11 is a chromatogram of a quantitation limit + detection limit solution detected under the conditions of example 2 in the present invention;
FIG. 12 is a chromatogram of a detection limiting solution detected under the conditions of example 2 in the present invention;
FIG. 13 is a chromatogram of a blank solution tested under the conditions of example 3 in accordance with the present invention;
FIG. 14 is a chromatogram of a system suitability solution tested under the conditions of example 3 in the present invention;
FIG. 15 is a chromatogram of a test solution tested under the conditions of example 3 in accordance with the present invention;
FIG. 16 is a chromatogram of a limiting quantitation solution detected under the conditions of example 3 in the present invention;
FIG. 17 is a chromatogram of a quantitation limit + detection limit solution detected under the conditions of example 3 in the present invention;
FIG. 18 is a chromatogram of a detection-limiting solution detected under the conditions of example 3 in the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention. The reagents and instruments used are not indicated by manufacturers, and conventional products can be obtained commercially.
The tofacitinib intermediate and the impurity reference substance used in the embodiment of the invention are prepared by the inventor.
Example 1
The chromatographic conditions were as follows:
a chromatographic column: agilent 5 TC-C18250 multiplied by 4.6mm
Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution
Mobile phase B: acetonitrile
Column temperature: 30 deg.C
Flow rate: 1.0ml/min
Detection wavelength: 210nm
Sample introduction amount: 5 μ l
The gradient elution procedure was:
TABLE 1 gradient elution procedure
| Time in minutes | Mobile phase A, volume% | Mobile phase B, |
| 0 | 95 | 5 |
| 5 | 95 | 5 |
| 55 | 30 | 70 |
| 56 | 95 | 5 |
| 70 | 95 | 5 |
Solution preparation:
impurity reference stock solution: precisely weighing about 25mg of each of the impurity A reference substance, the impurity B reference substance, the impurity C reference substance, the impurity D reference substance and the impurity E reference substance, placing the reference substances into a same 50ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution (pH7.0) -acetonitrile (volume ratio of 90:10) to dissolve and dilute the reference substances to scale, and shaking up to obtain the final product.
System applicability solution: taking about 50mg of the tofacitinib intermediate working reference substance, precisely weighing, placing into a 100ml measuring flask, precisely adding 1ml of the impurity reference substance stock solution into the 100ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio of 90:10) to dilute to scale, and shaking up to obtain the tofacitinib intermediate.
Test solution: taking about 50mg of tofacitinib intermediate, precisely weighing, placing in a 100ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dissolve and dilute to scale, and shaking up.
Tofacitinib intermediate control stock solutions: taking about 50mg of the tofacitinib intermediate working reference substance, precisely weighing, placing into a 100ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dissolve and dilute to scale, and shaking up to obtain the product.
Quantitative limiting solution: precisely measuring the impurity reference substance storage solution and the tofacitinib intermediate reference substance storage solution in measuring bottles of 1ml to 100ml respectively, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to a scale, shaking up, precisely measuring 1ml to 25ml, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to a scale, and shaking up to obtain the product, wherein the product is the quantitative limit solution of the impurity C, D, E, F and the main peak.
Quantitative limit + detection limit solution: precisely measuring 5ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to scale, and shaking up to obtain the quantitative limiting solution of the impurities A and B, which is also the detection limiting solution of the impurity C, D, E, F and the main peak.
Detection limiting solution: precisely measuring 5ml of the quantitative limit and detection limit solution, placing the solution into a 10ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to scale, and shaking up to obtain the detection limit solution of the impurities A and B.
And (3) determination: respectively injecting blank solution (namely diluent), system applicability solution, sample solution, quantitative limit + detection limit solution and detection limit solution into a high performance liquid chromatograph for detection, using octadecylsilane chemically bonded silica as a filler (the particle size is 5 mu m, the inner diameter of a column is 4.6mm, the length of the chromatographic column is 250mm), detecting according to the gradient elution program in the table 1, and recording a chromatogram.
Chromatograms of a blank solution (i.e., a diluent), a system applicability solution, a sample solution, a quantification limit + detection limit solution, and a detection limit solution are respectively shown in fig. 1, 2, 3, 4, 5, and 6, and it can be seen that fig. 1 shows that the blank does not interfere with the inspection of impurities; FIG. 2 shows that the separation degree between each impurity and tofacitinib intermediate is good, and the system applicability map data of the tofacitinib intermediate is shown in Table 2; FIG. 3 shows that impurities A, C and E are not detected in a self-made tofacitinib intermediate sample, and detected impurities B and D are below 0.5 wt%; the other single impurities detected are all below 0.5 wt%; fig. 4 and 5 show that the quantitation limits for tofacitinib intermediate and impurity a, impurity B, impurity C, impurity D, and impurity E are 0.04 wt%, 0.02 wt%, 0.04 wt%, and 0.04 wt%, respectively; FIGS. 5 and 6 show that the detection limits for tofacitinib intermediate and impurity A, impurity B, impurity C, impurity D and impurity E are 0.02 wt%, 0.01 wt%, 0.02 wt% and 0.02 wt%, respectively, below the limit of each impurity by 0.50 wt%; the method has high detection sensitivity.
Table 2 table of tofacitinib system applicability profiles in example 1
Example 2
The chromatographic conditions were as follows:
a chromatographic column: agilent 5 TC-C18250 multiplied by 4.6mm
Mobile phase A: 0.02mol/L potassium dihydrogen phosphate solution
Mobile phase B: acetonitrile
Column temperature: 30 deg.C
Flow rate: 1.0ml/min
Detection wavelength: 210nm
Sample introduction amount: 5 μ l
The gradient elution procedure was:
TABLE 3 gradient elution procedure
| Time in minutes | Mobile phase A, volume% | Mobile phase B, |
| 0 | 92 | 8 |
| 5 | 92 | 8 |
| 55 | 30 | 70 |
| 56 | 92 | 8 |
| 70 | 92 | 8 |
Solution preparation:
impurity reference stock solution: taking about 25mg of each of the impurity A reference substance, the impurity B reference substance, the impurity C reference substance, the impurity D reference substance and the impurity E reference substance, precisely weighing, placing in a same 50ml measuring flask, adding 0.02mol/L potassium dihydrogen phosphate solution (pH7.0) -acetonitrile (volume ratio of 90:10) to dissolve and dilute to scale, and shaking uniformly to obtain the final product.
System applicability solution: taking about 50mg of the tofacitinib intermediate working reference substance, precisely weighing, placing into a 100ml measuring flask, precisely adding 1ml of the impurity reference substance stock solution into the 100ml measuring flask, adding 0.02mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio of 90:10) to dilute to scale, and shaking up to obtain the tofacitinib intermediate.
Test solution: taking about 50mg of tofacitinib intermediate, precisely weighing, placing in a 100ml measuring flask, adding 0.02mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dissolve and dilute to scale, and shaking up.
Tofacitinib intermediate control stock solutions: taking about 50mg of the tofacitinib intermediate working reference substance, precisely weighing, placing into a 100ml measuring flask, adding 0.02mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dissolve and dilute to scale, and shaking up to obtain the product.
Quantitative limiting solution: precisely measuring the impurity reference substance storage solution and the tofacitinib intermediate reference substance storage solution in measuring bottles of 1ml to 100ml respectively, adding 0.02mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio of 90:10) to dilute to a scale, shaking up, precisely measuring 1ml to 25ml, adding 0.02mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio of 90:10) to dilute to a scale, and shaking up to obtain the product, wherein the product is the quantitative limit solution of the impurity C, D, E, F and the main peak.
Quantitative limit + detection limit solution: precisely measuring 5ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding 0.02mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to scale, and shaking up to obtain the quantitative limiting solution of the impurities A and B, which is also the detection limiting solution of the impurity C, D, E, F and the main peak.
Detection limiting solution: precisely measuring 5ml of the quantitative limit and detection limit solution, placing the solution into a 10ml measuring flask, adding 0.02mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to scale, and shaking up to obtain the detection limit solution of the impurities A and B.
And (3) determination: respectively injecting blank solution (namely diluent), system applicability solution, sample solution, quantitative limit + detection limit solution and detection limit solution into a high performance liquid chromatograph for detection, using octadecylsilane chemically bonded silica as a filler (the particle size is 5 mu m, the inner diameter of a column is 4.6mm, the length of the chromatographic column is 250mm), detecting according to a gradient elution program shown in Table 3, and recording a chromatogram.
Chromatograms of a blank solution (i.e., a diluent), a system applicability solution, a sample solution, a limit of quantitation + limit of detection solution, and a limit of detection solution are shown in fig. 7, 8, 9, 10, 11, and 12, respectively, and it can be seen that fig. 7 shows that blanks do not interfere with impurity inspection; FIG. 8 shows that the separation between each impurity and tofacitinib intermediate is good, and specific tofacitinib intermediate system applicability profile data are shown in Table 4; FIG. 9 shows that impurities A, C and E are not detected in the home-made tofacitinib intermediate sample, and the detected impurities B and D are below 0.5 wt%; the other single impurities detected are all below 0.5 wt%; fig. 10 and 11 show that the quantitation limits for tofacitinib intermediate and impurity a, impurity B, impurity C, impurity D, and impurity E are 0.04 wt%, 0.02 wt%, 0.04 wt%, and 0.04 wt%, respectively; FIGS. 11 and 12 show that the detection limits for tofacitinib intermediate and impurity A, impurity B, impurity C, impurity D, and impurity E are 0.02 wt%, 0.01 wt%, 0.02 wt%, and 0.02 wt%, respectively, below the limit of each impurity by 0.50 wt%; the method has high detection sensitivity.
Table 4 tofacitinib system applicability profile data table in example 2
Example 3
The chromatographic conditions were as follows:
a chromatographic column: agilent 5 TC-C18250 multiplied by 4.6mm
Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution
Mobile phase B: acetonitrile-methanol (volume ratio 60:40)
Column temperature: 30 deg.C
Flow rate: 1.0ml/min
Detection wavelength: 210nm
Sample introduction amount: 5 μ l
The gradient elution procedure was:
TABLE 5 gradient elution procedure
| Time in minutes | Mobile phase A, volume% | Mobile phase B, |
| 0 | 90 | 10 |
| 5 | 90 | 10 |
| 55 | 30 | 70 |
| 56 | 90 | 10 |
| 70 | 90 | 10 |
Solution preparation:
impurity reference stock solution: precisely weighing about 25mg of each of the impurity A reference substance, the impurity B reference substance, the impurity C reference substance, the impurity D reference substance and the impurity E reference substance, placing the reference substances into a same 50ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution (pH7.0) -acetonitrile (volume ratio of 90:10) to dissolve and dilute the reference substances to scale, and shaking up to obtain the final product.
System applicability solution: taking about 50mg of the tofacitinib intermediate working reference substance, precisely weighing, placing into a 100ml measuring flask, precisely adding 1ml of the impurity reference substance stock solution into the 100ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio of 90:10) to dilute to scale, and shaking up to obtain the tofacitinib intermediate.
Test solution: taking about 50mg of tofacitinib intermediate, precisely weighing, placing in a 100ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dissolve and dilute to scale, and shaking up.
Tofacitinib intermediate control stock solutions: taking about 50mg of the tofacitinib intermediate working reference substance, precisely weighing, placing into a 100ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dissolve and dilute to scale, and shaking up to obtain the product.
Quantitative limiting solution: precisely measuring the impurity reference substance storage solution and the tofacitinib intermediate reference substance storage solution in measuring bottles of 1ml to 100ml respectively, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to a scale, shaking up, precisely measuring 1ml to 25ml, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to a scale, and shaking up to obtain the product, wherein the product is the quantitative limit solution of the impurity C, D, E, F and the main peak.
Quantitative limit + detection limit solution: precisely measuring 5ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to scale, and shaking up to obtain the quantitative limiting solution of the impurities A and B, which is also the detection limiting solution of the impurity C, D, E, F and the main peak.
Detection limiting solution: precisely measuring 5ml of the quantitative limit and detection limit solution, placing the solution into a 10ml measuring flask, adding 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (volume ratio is 90:10) to dilute to scale, and shaking up to obtain the detection limit solution of the impurities A and B.
And (3) determination: respectively injecting blank solution (namely diluent), system applicability solution, sample solution, quantitative limit + detection limit solution and detection limit solution into a high performance liquid chromatograph for detection, using octadecylsilane chemically bonded silica as a filler (the particle size is 5 mu m, the inner diameter of a column is 4.6mm, the length of the chromatographic column is 250mm), detecting according to a gradient elution program shown in Table 3, and recording a chromatogram.
Chromatograms of a blank solution (i.e., a diluent), a system applicability solution, a sample solution, a limit of quantitation + limit of detection solution, and a limit of detection solution are shown in fig. 13, 14, 15, 16, 17, and 18, respectively, and it can be seen that fig. 13 shows that the blank does not interfere with the inspection of impurities; FIG. 14 shows that the separation between each impurity and tofacitinib intermediate is good, and specific tofacitinib intermediate system applicability profile data are shown in Table 6; FIG. 15 shows that impurities A, C and E are not detected in the home-made tofacitinib intermediate sample, and that impurities B and D are below 0.5 wt%; the other single impurities detected are all below 0.5 wt%; fig. 16 and 17 show that the quantitation limits for tofacitinib intermediate and impurity a, impurity B, impurity C, impurity D, and impurity E are 0.04 wt%, 0.02 wt%, 0.04 wt%, and 0.04 wt%, respectively; fig. 17 and 18 show that the detection limits of tofacitinib intermediate and impurity a, impurity B, impurity C, impurity D, and impurity E are 0.02 wt%, 0.01 wt%, 0.02 wt%, and 0.02 wt%, respectively, below the limit of each impurity by 0.50 wt%; the method has high detection sensitivity.
Table 6 tofacitinib system applicability profiles data table in example 3
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (10)
1. A method for separating and measuring related substances of a tofacitinib intermediate by using HPLC (high performance liquid chromatography), which is characterized by comprising the following steps: the method comprises the following steps: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, using 0.005-0.03 mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile, methanol or acetonitrile-methanol as a mobile phase B, wherein the flow rate is 0.8-1.2 ml/min, the column temperature is 25-40 ℃, and the volume ratio of the acetonitrile to the methanol is 40: 60-60: 40; and detecting related substances of the tofacitinib intermediate by adopting an ultraviolet detector, wherein the detection wavelength of the ultraviolet detector is 205-230 nm.
2. The method for the determination of tofacitinib intermediate related substances by HPLC separation according to claim 1, characterized in that: the conditions of the gradient elution are as follows:
。
3. The method for the determination of tofacitinib intermediate related substances by HPLC separation according to claim 1, characterized in that: the conditions of the gradient elution are as follows:
。
4. The method for the determination of tofacitinib intermediate related substances by HPLC separation according to claim 1, characterized in that: the mobile phase A is 0.01mol/L potassium dihydrogen phosphate solution, and the mobile phase B is acetonitrile.
5. The method for the determination of tofacitinib intermediate related substances by HPLC separation according to claim 1, characterized in that: the detection wavelength of the ultraviolet detector is 210 nm.
6. The method for the determination of tofacitinib intermediate related substances by HPLC separation according to claim 1, characterized in that: the related substances of the tofacitinib intermediate comprise one or more of impurities A, B, C, D and E, and the specific structural formula is as follows:
7. the method for the determination of tofacitinib intermediate related substances by HPLC separation according to claim 1, characterized in that: the length of the chromatographic column is 150 mm-250 mm; the particle size of the filler is 1.8-5 μm.
8. The method for the determination of tofacitinib intermediate related substances by HPLC separation according to claim 1, characterized in that: the column length was 250 mm.
9. The method for the determination of tofacitinib intermediate related substances by HPLC separation according to claim 1, characterized in that: the particle size of the filler is 5 mu m.
10. The method for the determination of tofacitinib intermediate related substances by HPLC separation according to any one of claims 1 to 9, wherein: the flow rate was 1.0ml/min and the column temperature was 30 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010739267.8A CN111983056B (en) | 2020-07-28 | 2020-07-28 | Method for separating and measuring related substances of tofacitinib intermediate by using HPLC (high performance liquid chromatography) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010739267.8A CN111983056B (en) | 2020-07-28 | 2020-07-28 | Method for separating and measuring related substances of tofacitinib intermediate by using HPLC (high performance liquid chromatography) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111983056A true CN111983056A (en) | 2020-11-24 |
| CN111983056B CN111983056B (en) | 2022-05-03 |
Family
ID=73444691
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010739267.8A Active CN111983056B (en) | 2020-07-28 | 2020-07-28 | Method for separating and measuring related substances of tofacitinib intermediate by using HPLC (high performance liquid chromatography) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111983056B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115236261A (en) * | 2022-05-10 | 2022-10-25 | 汉瑞药业(荆门)有限公司 | HPLC-UV detection method for purity of tofacitinib intermediate |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104860950A (en) * | 2014-02-24 | 2015-08-26 | 重庆医药工业研究院有限责任公司 | Method used for preparing 4-chloropyrrolo[2,3-d]pyrimidine |
| CN105294698A (en) * | 2015-11-30 | 2016-02-03 | 山东省药学科学院 | Crystalline form of N-methyl-N-((3R,4R)-4-methyl-1-benzyl-3-piperidyl)-7H-pyrrolo[2,3-D]pyrimidine-4-amine |
| US20160122354A1 (en) * | 2013-06-05 | 2016-05-05 | Srinivasan Thirumalai Rajan | PROCESS FOR THE PREPARATION OF (3R,4R)-4-METHYL-3-(METHYL-7H-PYRROLO[2,3-D]PYRIMIDIN-4-YL-AMINO)-ß-OXO-1-PIPERIDINEPROPANENITRILE AND ITS SALTS |
| CN109879879A (en) * | 2019-03-25 | 2019-06-14 | 国药集团容生制药有限公司 | A kind of support method replaces the preparation process of cloth intermediate |
-
2020
- 2020-07-28 CN CN202010739267.8A patent/CN111983056B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160122354A1 (en) * | 2013-06-05 | 2016-05-05 | Srinivasan Thirumalai Rajan | PROCESS FOR THE PREPARATION OF (3R,4R)-4-METHYL-3-(METHYL-7H-PYRROLO[2,3-D]PYRIMIDIN-4-YL-AMINO)-ß-OXO-1-PIPERIDINEPROPANENITRILE AND ITS SALTS |
| CN104860950A (en) * | 2014-02-24 | 2015-08-26 | 重庆医药工业研究院有限责任公司 | Method used for preparing 4-chloropyrrolo[2,3-d]pyrimidine |
| CN105294698A (en) * | 2015-11-30 | 2016-02-03 | 山东省药学科学院 | Crystalline form of N-methyl-N-((3R,4R)-4-methyl-1-benzyl-3-piperidyl)-7H-pyrrolo[2,3-D]pyrimidine-4-amine |
| CN109879879A (en) * | 2019-03-25 | 2019-06-14 | 国药集团容生制药有限公司 | A kind of support method replaces the preparation process of cloth intermediate |
Non-Patent Citations (2)
| Title |
|---|
| XIAO WU 等: "Identification of related substances in tofacitinib citrate by LC-MS techniques for synthetic process optimization", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| 张仲奎 等: "Tofacitinib的合成", 《中国医药工业杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115236261A (en) * | 2022-05-10 | 2022-10-25 | 汉瑞药业(荆门)有限公司 | HPLC-UV detection method for purity of tofacitinib intermediate |
| CN115236261B (en) * | 2022-05-10 | 2024-04-19 | 武汉海特生物创新医药研究有限公司 | HPLC-UV detection method for tofacitinib intermediate purity |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111983056B (en) | 2022-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110441449B (en) | Method for detecting related substances in fasudil hydrochloride raw material or injection | |
| CN109374784B (en) | Method for separating and measuring related substances of dapagliflozin bulk drug by using HPLC (high performance liquid chromatography) | |
| CN114264747A (en) | High performance liquid chromatography detection method for empagliflozin intermediate and related substances thereof | |
| CN107064350A (en) | Method for detecting suspected genotoxic impurity of tofacitinib citrate | |
| CN115097023A (en) | High performance liquid chromatography detection method for zolmitamine related substances | |
| CN111983055B (en) | Method for separating and measuring rivaroxaban intermediate related substances by using HPLC (high performance liquid chromatography) | |
| CN109632981A (en) | A kind of method of LC-MS detection 2,2,6,6- tetramethyl piperidine nitrogen oxides | |
| CN111999400B (en) | Method for separating and determining impurities of bulk drugs of baricitinib by using HPLC (high performance liquid chromatography) | |
| CN111983056B (en) | Method for separating and measuring related substances of tofacitinib intermediate by using HPLC (high performance liquid chromatography) | |
| CN108699018B (en) | Method for analyzing and distinguishing preparations of dianhydrogalactitol and derivatives or analogs thereof | |
| CN111239286B (en) | Method for detecting genotoxic impurities in tenofovir | |
| CN109374782B (en) | Method for separating and measuring related substances of empagliflozin bulk drug by using HPLC (high performance liquid chromatography) | |
| CN111624275A (en) | Method for measuring related substances of compound ipratropium bromide solution for inhalation by high performance liquid chromatography | |
| CN111983054B (en) | Method for separating and measuring related substances of empagliflozin intermediate by using HPLC (high performance liquid chromatography) | |
| CN114544850B (en) | Dapagliflozin intermediate and impurity detection method | |
| CN110606846A (en) | Tofacitinib citrate impurity and analysis method and application thereof | |
| CN115684397A (en) | Method for determining content of genotoxic impurity hydroxylamine hydrochloride in parecoxib | |
| CN110836930A (en) | Method for measuring content of dichlorobutane in levetiracetam by gas chromatography-mass spectrometry | |
| CN109738536B (en) | Method for separating benzaldehyde and nitrobenzaldehyde by using high performance liquid chromatography | |
| CN111458418B (en) | Method for detecting residual ammonium in enoxaparin sodium | |
| CN115774061A (en) | Method for detecting acetic acid in 1-cyclohexyl piperazine | |
| CN111257440A (en) | GC-HS-based method for determining potential genotoxic impurities in sodium valproate | |
| CN109374783B (en) | Method for separating and determining related substances of canagliflozin bulk drug by using HPLC (high performance liquid chromatography) | |
| CN115856141B (en) | Method for separating and measuring tinib key intermediate and relevant impurities thereof by HPLC | |
| CN115453014B (en) | Detection method of butylphthalide and related substances thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |