CN114252513A - Method for detecting and analyzing impurities in tofacitinib citrate - Google Patents
Method for detecting and analyzing impurities in tofacitinib citrate Download PDFInfo
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The process of the invention is shown to be superior by comparing example 1 (inventive process) with example 2 (imported drug registration standard). The invention discloses a method for detecting and analyzing impurities in tofacitinib citrate, which detects 13 impurities in tofacitinib citrate by optimizing chromatographic conditions and adopting a high performance liquid chromatography, improves the detection means of the quality control of the tofacitinib citrate product, and simultaneously improves the safety of the product.
Description
Technical Field
The invention belongs to the field of new medicine technology, and relates to an analysis and determination method for 13 impurities (degradation impurities and process impurities) in tofacitinib citrate. In particular to a high performance liquid chromatography, which adopts an ultraviolet detector and selects a proper mobile phase and a chromatographic column to quantitatively analyze 13 impurities in tofacitinib citrate.
Background
Tofacitinib citrate (tofacitinib citrate), chemically known as 3- [ (3R, 4R) -4-methyl-3- [ methyl- (7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) amino ] piperidin-1-yl ] -3-oxopropanenitrile citrate chemical book salt, is a novel Janus kinase inhibitor developed by the american pfeiri company, approved by the american FDA for marketing at 11 months 2012, under the trade name Xeljanz. The product can be used in combination with methotrexate for treating active rheumatoid arthritis with insufficient response to TNF (tumor necrosis factor) inhibitor. In order to better control the quality of tofacitinib citrate and improve the safety of the medicament, impurities (including degradation impurities and process impurities) possibly generated in the tofacitinib citrate synthesis process need to be strictly monitored, and the common impurities in tofacitinib citrate have the following structural formula:
although the existing tofacitinib citrate tablet import medicine registration standard (JX20130251) exists at present, a related substance detection method in the standard is an ultrahigh liquid chromatography, the cost is high, the applicability is low, the method is high in diluent interference and low in detection capability, the detection requirement of tofacitinib citrate cannot be met, the quality of tofacitinib citrate cannot be effectively controlled, and the safety of medicines is improved. Therefore, a method with wide application range, low cost and simple operation is needed to be developed for quality control.
Disclosure of Invention
The invention aims to provide a method for detecting 13 impurities in tofacitinib citrate, which has the advantages of good stability, good reproducibility, simple operation and high precision. The method for determining 13 impurities in tofacitinib citrate is convenient and feasible, has high accuracy, can effectively control the quality of tofacitinib citrate, and improves the safety of medicaments.
The technical scheme adopted by the invention for solving the technical problems is as follows: the method for detecting and analyzing the impurities in tofacitinib citrate comprises the steps of adopting a common high performance liquid chromatography, wherein the sample injection volume is 5-100 mu l, utilizing an ultraviolet detector, the detection wavelength is 210-350 nm, the flow rate of a mobile phase is 0.3-2.0 ml/min, an alkyl bonded silica gel chromatographic column is a stationary phase, the column temperature is 20-30 ℃, and the mobile phase is: taking an ammonium formate solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution on the mobile phase in the following way:
0-60 min, the proportion of the mobile phase A is 80-90%, and the proportion of the mobile phase B is 10-20%;
the proportion of the mobile phase A is changed into 40-60% and the proportion of the mobile phase B is changed into 40-60% in 60-70 min;
71-80 min, wherein the proportion of the mobile phase A is 80-90%; the proportion of the mobile phase B becomes 10 to 20 percent.
The impurities in tofacitinib citrate are 13 as follows:
in some embodiments of the invention, the mobile phase A is 2-5 g/L ammonium formate solution, and in some specific embodiments, the mobile phase A is 3g/L ammonium formate solution.
In some embodiments, the ammonium formate solution has a pH of 3 to 5, and in a specific embodiment, a pH of 4. The pH can be adjusted by methods customary in the art, for example by using formic acid.
According to the detection method, when tofacitinib citrate is detected, a solution containing 0.2 mg-1 mg of tofacitinib citrate in every 1ml is dissolved and diluted by a solvent and is used as a test solution to be injected into a high performance liquid chromatograph. On the basis of the technical scheme, the tofacitinib citrate is dissolved and diluted by a solvent into a solution containing 0.2 mg-1 mg of tofacitinib citrate in each 1ml of the solution to be used as a test solution. The solvent is mobile phase and water, and is preferably mobile phase; the concentration of the test solution is preferably 0.7 mg/ml.
On the basis of the technical scheme, the flow velocity of the mobile phase is 0.5-1.5 ml/min, and the best flow velocity is 1.0 ml/min.
On the basis of the technical scheme, the sample injection volume is 5-50 mu l, and the optimal sample injection volume is 20 mu l.
On the basis of the technical scheme, the stationary phase alkyl bonded silica gel chromatographic column is preferably an octadecyl bonded silica gel chromatographic column.
On the basis of the technical scheme, the detection wavelength is 210 nm-350 nm, and the optimal detection wavelength is 289 nm.
In some embodiments of the invention, the mobile phase is subjected to gradient elution in the following manner:
0-60 min, wherein the proportion of the mobile phase A is 85% -90%, and the proportion of the mobile phase B is 10% -15%;
the proportion of the mobile phase A is changed to 48-52 percent and the proportion of the mobile phase B is changed to 48-52 percent in 60-70 min;
71-80 min, wherein the proportion of the mobile phase A is 85% -90%; the proportion of the mobile phase B becomes 10 to 15 percent.
In one embodiment of the invention, the mobile phase is subjected to gradient elution in the following manner:
the invention has the beneficial effects that: the method utilizes convenient and quick high performance liquid chromatography to detect 13 impurities in tofacitinib citrate, improves the detection means of the quality control of the tofacitinib citrate product, and simultaneously improves the safety of the product.
Drawings
FIG. 1 is a blank solvent chromatogram;
FIG. 2 is a chromatogram of a sample mixed with tofacitinib citrate and 13 impurities;
FIG. 3 is a tofacitinib citrate sample map;
FIG. 4 is a blank solvent chromatogram of the method for registering substances relevant to the standard of import drugs;
FIG. 5 is a sample map of related substances of imported drug registration standard
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.
The instrument comprises the following steps: the high performance liquid chromatograph comprises a liquid phase pump, an ultraviolet detector and a sample injector
Reagent: ammonium formate (analytical grade), formic acid (analytical grade), acetonitrile
A chromatographic column: octadecyl bonded silica gel chromatographic column
Example 1:
the determination is carried out by high performance liquid chromatography (China pharmacopoeia 2015 edition of general rules 0512).
Chromatographic conditions and system applicability test using octadecylsilane chemically bonded silica as filler (Amethyl C18-H, 250mm × 4.6mm, 5 μm or equivalent performance chromatographic column); taking an ammonium formate solution (taking 3.0g of ammonium formate, adding water for dissolving, diluting to 1000ml, adjusting the pH value to 4.0 by using formic acid) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out linear gradient elution according to the table 1; the flow rate was 1.0ml per minute; the detection wavelength is 289 nm; the column temperature was 25 ℃. And (2) taking 20 mu l of system applicability solution, injecting the solution into a liquid chromatograph, recording a chromatogram, wherein the appearance sequence sequentially comprises impurities MPPA (impurity A), TFA (impurity), TFBA (impurity), WHE1305-ZE (impurity E), HTFCS (impurity C), TFPE (impurity), tofacitinib, D-TFTN (impurity B), WHE1305-ZF (impurity F), TFZZ (impurity D), CBPPA (impurity), CTFCS (impurity), TFPB (impurity) and TFCA (impurity TFCA), the number of theoretical plates is not less than 5000 according to tofacitinib peaks, and the separation degree between each component peak is not less than 1.5.
TABLE 1 gradient elution procedure
Measuring to obtain 17.5mg of the product, placing in a 25ml measuring flask, adding about 15ml of diluent [ ammonium formate solution (3.0 g of ammonium formate is taken, water is added for dissolving and diluting to 1000ml, pH value is adjusted to 4.0 by formic acid) -acetonitrile (88:12) ] for dissolving by ultrasonic wave, diluting to scale by diluent, shaking up to obtain sample solution; precisely measuring 1ml, placing in a 100ml measuring flask, diluting to scale with diluent, and shaking to obtain a control solution; taking an impurity MPPA (impurity A) reference substance, an impurity TFA reference substance, an impurity TFBA reference substance, an impurity WHE1305-ZE (impurity E) reference substance, an impurity HTFCS (impurity C) reference substance, an impurity TFPE reference substance, an impurity D-TFTN (impurity B) reference substance, an impurity TFZZ (impurity D) reference substance, an impurity CBPPA reference substance, an impurity CTFCS reference substance, an impurity TFPB reference substance and an impurity TFCA reference substance which are respectively 17.5mg, respectively placing the impurities into different 100ml measuring bottles, adding a proper amount of methanol for ultrasonic dissolution, diluting the impurities to scale by using the methanol, shaking the impurities uniformly to serve as storage solutions of the impurity reference substances; placing 17.5mg of impurity WHE1305-ZF (impurity F) reference substance into a 100ml measuring flask, adding proper amount of dimethyl sulfoxide for ultrasonic dissolution, diluting with acetonitrile-water (50:50) to scale, shaking up to obtain impurity F reference substance stock solution; taking about 17.5mg of tofacitinib citrate as a reference substance, placing the tofacitinib citrate in a 25ml measuring flask, adding about 15ml of diluent for ultrasonic dissolution, respectively adding 0.25ml of each impurity stock solution, diluting to the scale with the diluent, shaking up, and taking the solution as a system applicability solution. Precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If an impurity peak exists in a chromatogram of a test solution, the peak with relative retention time less than 0.3 is ignored, and the content of the impurity is calculated according to a main component self-comparison method multiplied by a correction factor, which all accords with the corresponding limit regulation in the table 2.
Table 2 known impurity limits
As a result, as shown in FIGS. 1 to 3, it can be seen that the method of the present invention can achieve a good separation of impurities in 13.
Comparative example 1:
reference is made to imported drug registration standards.
Chromatographic conditions are as follows: chromatographic column (Acquity BEH UPLC C18, 100mm × 2.1mm, 1.7 μm or equivalent performance chromatographic column) using ultra-high liquid chromatograph and octadecylsilane chemically bonded silica as filler; at 10mM KH2PO4The solution (pH adjusted to 6.8 with NaOH solution) -acetonitrile (92:8) as mobile phase A and acetonitrile as mobile phase B, linear gradient elution was performed as per Table 3; the flow rate is 0.3ml per minute (if necessary, the flow rate can be properly adjusted to keep the retention time of the tofacitinib peak within the range of 5-9 minutes); the detection wavelength is 210 nm; the column temperature was 65 ℃. The injection volume was 2. mu.l.
TABLE 3 gradient elution procedure
As a result, as shown in fig. 4 and 5, good separation of the impurities in 13 cannot be achieved. And the blank solution has large interference and low detection capability.
Claims (10)
1. The method for detecting and analyzing impurities in tofacitinib citrate is characterized by comprising the following steps of adopting a high performance liquid chromatography, wherein the sample injection volume is 5-100 mu l, utilizing an ultraviolet detector, detecting the wavelength of 210-350 nm, the flow rate of a mobile phase is 0.3-2.0 ml/min, using an alkyl bonded silica gel chromatographic column as a stationary phase, and the column temperature is 20-30 ℃, and using the mobile phase: taking an ammonium formate solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution on the mobile phase in the following way:
0-60 min, the proportion of the mobile phase A is 80-90%, and the proportion of the mobile phase B is 10-20%;
the proportion of the mobile phase A is changed into 40-60% and the proportion of the mobile phase B is changed into 40-60% in 60-70 min;
71-80 min, wherein the proportion of the mobile phase A is 80-90%; the proportion of the mobile phase B becomes 10 to 20 percent.
2. The assay method according to claim 1, wherein the impurities in tofacitinib citrate are 13 of the following types:
3. the analytical method according to claim 1, wherein the mobile phase A is 2 to 5g/L ammonium formate solution, preferably 3g/L ammonium formate solution.
4. The analytical method according to claim 1, wherein the ammonium formate solution has a pH of 3 to 5, preferably a pH of 4.
5. The analysis method of claim 1, wherein in the detection method, the tofacitinib citrate is dissolved and diluted by a solvent to a solution containing 0.2mg to 1mg of tofacitinib citrate in each 1ml as a test solution; preferably, the tofacitinib citrate is dissolved and diluted by a solvent to form a solution containing 0.7mg of tofacitinib citrate in every 1ml of the solution as a test solution; preferably, the solvent is a mobile phase, water, most preferably a mobile phase.
6. The assay of claim 1, wherein the mobile phase flow rate is 1.0 ml/min.
7. The method according to claim 1, wherein the sample volume is 5 to 50 μ l, preferably 20 μ l.
8. The analytical method of claim 1, wherein the stationary phase is an octadecyl-bonded silica chromatographic column.
9. The assay of claim 1, wherein the detection wavelength is 289 nm.
10. The assay of claim 1, wherein the mobile phase is subjected to gradient elution by:
0-60 min, wherein the proportion of the mobile phase A is 85% -90%, and the proportion of the mobile phase B is 10% -15%;
the proportion of the mobile phase A is changed to 48-52 percent and the proportion of the mobile phase B is changed to 48-52 percent in 60-70 min;
71-80 min, wherein the proportion of the mobile phase A is 85% -90%; the proportion of the mobile phase B is changed into 10 to 15 percent;
preferably, the mobile phase is subjected to gradient elution in the following manner:
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| CN118010891A (en) * | 2024-04-09 | 2024-05-10 | 深圳市瑞华制药技术有限公司 | Detection and analysis method for related substances in tofacitinib citrate sustained release tablet |
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| CN118010891A (en) * | 2024-04-09 | 2024-05-10 | 深圳市瑞华制药技术有限公司 | Detection and analysis method for related substances in tofacitinib citrate sustained release tablet |
| CN118010891B (en) * | 2024-04-09 | 2024-06-04 | 深圳市瑞华制药技术有限公司 | Detection and analysis method for related substances in tofacitinib citrate sustained release tablet |
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