CN117783358A - Method for separating and measuring critical starting materials and related impurities of ritatist by HPLC (high Performance liquid chromatography) - Google Patents
Method for separating and measuring critical starting materials and related impurities of ritatist by HPLC (high Performance liquid chromatography) Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention belongs to the field of analytical chemistry, and in particular relates to a method for separating and measuring a critical starting material of ritustilast and related impurities thereof by an HPLC method. The method can effectively separate the key starting materials of the rituximab and related impurities thereof, has high sensitivity and separation degree, good repeatability and durability, simple operation and stable and reliable result, and has extremely important significance for realizing the quality control of the rituximab medicament.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for detecting a critical starting material of ritatist, and particularly relates to an HPLC method for separating and measuring the critical starting material of ritatist and related impurities thereof.
Background
Lifiteglast (Lifiteglast) is a novel small molecule integrin inhibitor that reduces T lymphocyte-mediated inflammation levels by inhibiting the binding of lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1. Litaset was developed by Ireland Xial development (Shire Dev Llc), 7 in 2016, and was approved by the FDA for the treatment of dry eye symptoms and signs. Litaset is a prescription drug for treating dry eye symptoms and symptoms by inhibiting inflammation caused by dry eye, which can treat the eye injury caused by dry eye and relieve discomfort symptoms caused by dry eye.
Generally, a drug should have a total impurity content of less than 1.0% and a single impurity content of less than 0.1%, and strict control is required for impurities generated during the preparation of rituximab or related substances introduced into the preparation, both in the drug substance and in the preparation. 2- (tert-butyloxycarbonyl) -5, 7-dichloro-1, 2,3, 4-tetrahydroisoquinoline-6-carboxylic acid is a key starting material of the rituximab, quality control of the compound plays an important role in preparing high-quality rituximab, and detection of related substances in the rituximab and preparations is mainly carried out in the prior art, so that detection reports on related substances in the rituximab starting material are less. Therefore, development of an efficient and accurate analysis method is needed, and mass analysis of key starting materials of the pentagaster drug is important for subsequent preparation of high-purity pentagaster products.
Disclosure of Invention
Accordingly, the present invention is directed to a method for separating and determining key starting materials of rituximab and related impurities by HPLC; the method can effectively separate the key starting materials of the rituximab and related impurities thereof, has high sensitivity and separation degree, good repeatability and durability, simple operation and stable and reliable result.
In order to achieve the above object, the applicant has determined, through a large number of experiments, that the technical scheme of the present invention is:
the method for separating and measuring the key starting materials of the ritatist and related impurities by an HPLC method comprises the steps that a chromatographic column is adopted, octadecylsilane chemically bonded silica is used as a filler, a mobile phase A and a mobile phase B are adopted for gradient elution, and the gradient elution enters a detector for detection; the related impurities comprise one or more of impurities A, B, C, D, E and F, and the specific structural formula is shown as follows:
the mobile phase A is a mixed solvent of phosphate buffer solution and an organic solvent, and the mobile phase B is an organic solvent.
The key starting material of the rituximab is 2- (tert-butoxycarbonyl) -5, 7-dichloro-1, 2,3, 4-tetrahydroisoquinoline-6-carboxylic acid, which is the key starting material for synthesizing the rituximab and has a chemical formula of C 15 H 17 Cl 2 NO 4 The structural formula is shown as the following formula (I), and the invention is also called as a compound of the formula I or a key starting material of the litazost:
further, the mobile phase A is perchloric acid aqueous solution;
further, the perchloric acid proportion in the perchloric acid aqueous solution is 0.1% -0.2%;
preferably, the perchloric acid proportion in the perchloric acid aqueous solution is 0.15%;
further, the mobile phase B is one or more of acetonitrile, ethanol and methanol;
preferably, the mobile phase B is acetonitrile.
The flow rate at which the mobile phase is eluted is 0.8-1.2ml/min, preferably 1.0 ml/min.
The gradient elution settings were as follows:
as a preference, the gradient elution is set as follows:
further, the filler particles of the octadecylsilane chemically bonded silica have a particle size of 2 to 5 μm; the column temperature of the chromatographic column is 25-35 ℃.
As one preference, the filler particles of the octadecylsilane chemically bonded silica have a particle size of 5 μm; the column temperature of the chromatographic column was 30 ℃.
Further, the detection wavelength of the detector is 220+/-5 nm.
Preferably, the detection wavelength of the detector is 220nm.
Further, the method for separating and measuring the key starting materials of the rituximab and related impurities thereof by an HPLC method comprises the following steps:
1) Preparing a test sample solution: taking a proper amount of critical starting materials of the ritodynamic, precisely weighing, adding 2/5 volume of methanol for ultrasonic dissolution, and diluting with water to prepare a solution containing about 0.3mg per 1 ml;
2) Preparing a control solution: taking a proper amount of test solution, and diluting with a diluent to prepare a solution with about 0.3 mug in each 1ml serving as a control solution;
3) Preparing a system applicability solution: taking a critical starting material of rituximab and impurities A, B, C, D, E and F thereof, and dissolving and diluting the critical starting material with a diluent to prepare a system applicability solution;
4) Taking the solution with the suitability of the system in the step 3), carrying out high performance liquid chromatography analysis, recording a chromatogram, determining the retention time of the critical starting materials of the litaxest and the impurities, taking the sample solution in the step 1) and the control solution in the step 2) respectively, and calculating the content of the impurities in the critical starting materials of the litaxest in the sample solution according to a main component self-control method.
5) The linear correlation coefficients of the key starting materials of the rituximab and the impurities A, B, C, D, E and F are shown in the following table:
the diluent is a methanol-water (volume ratio of 40:60) solution.
The second object of the invention is to provide a reagent composition for solid-liquid separation determination of critical starting materials of ritatist and impurities thereof, which consists of the following reagents:
reagent A: aqueous perchloric acid;
reagent B: an organic solvent;
the related impurities comprise one or more of impurities A, B, C, D, E and F;
the proportion of perchloric acid in the perchloric acid aqueous solution is 0.1% -0.2%; the organic solvent is one or more of acetonitrile, ethanol and methanol.
Preferably, the perchloric acid proportion in the perchloric acid aqueous solution is 0.15%; the organic solvent is acetonitrile.
The reagent composition for solid-liquid separation determination of the critical starting materials and the impurities thereof provided by the invention can effectively separate the critical starting materials and the impurities thereof, and has extremely important significance for realizing the quality control of the critical starting materials and the properties of the critical starting materials.
The invention has the beneficial effects that:
1) The invention provides a method for separating and measuring the key starting materials of the rituximab and related impurities thereof by an HPLC method, the method can effectively separate the key starting materials of the rituximab and related impurities thereof, and the method has high sensitivity and separation degree, good repeatability and durability, simple operation and stable and reliable results.
2) According to research, the impurities B and C of the key starting material in the formula I are very close to the structure of the key starting material, if the structure is not controlled in the step, the impurities are difficult to remove if the reaction is carried into the bulk drug continuously, so that each impurity needs to be strictly monitored in the step of reaction, namely, the analysis research of the key starting material of the rituximab plays a key role in controlling the synthesis reaction and improving the quality of the key starting material of the rituximab, the quality of the finished product of the rituximab is directly influenced, and the method has extremely important significance in realizing the quality control of the key starting material of the rituximab and the rituximab.
Drawings
FIG. 1 shows a chromatogram of a solution of the system suitable for use under the conditions of the inventive example, with the sequence of peaks given by impurity A (retention time Rt21.748 minutes), impurity B (retention time Rt 13.230 minutes), compound of formula I (retention time Rt 20.747), impurity C (retention time Rt 5.875 minutes), impurity D (retention time Rt 27.681 minutes), impurity E (retention time Rt 12.329 minutes), impurity F (retention time Rt 24.129 minutes);
FIG. 2 is a chromatogram of a system applicability solution under the conditions of comparative example 1;
FIG. 3 is a chromatogram of a system applicability solution under the conditions of comparative example 2;
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Experimental methods without specific conditions noted in the preferred embodiments, the present invention is better illustrated by the examples according to the conventional conditions, but the present invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention. The key starting materials for rituximab in this example refer to compounds of formula I.
Example 1
1 chromatographic conditions:
octadecylsilane chemically bonded silica is used as filler (Welch ultimate LP-C18, 250 mm. Times.4.6 mm,5 μm is suitable); taking 0.15% perchloric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; linear gradient elution was performed as follows; the column temperature is 30 ℃; the flow rate is 1.0ml per minute; the detection wavelength is 220nm; the sample volume was 10. Mu.l.
2 methods and results
2.1 preparation of solutions
Taking a proper amount of critical starting materials of the litaxetil, precisely weighing, adding 2/5 volume of methanol for ultrasonic dissolution, and quantitatively diluting with water to prepare a solution containing about 0.3mg per 1ml as a test solution; a suitable amount of the sample solution was precisely measured, and diluted with a diluent to prepare a solution containing about 0.3. Mu.g per 1ml as a control solution.
2.2 specificity
The preparation method comprises the steps of taking a right amount of each of critical starting materials of the rituximab, the impurities B, the impurities C, the impurities D, the impurities E and the impurities F, precisely weighing, placing the materials into a same measuring flask, adding a diluent to dissolve and dilute the materials to prepare a solution containing about 0.3mg of a compound of the formula I and about 0.3 mug of each impurity per 1ml of the solution, and taking the solution as a system applicability solution. 10 μl of the solution was precisely measured and injected into a liquid chromatograph, the chromatogram was recorded, and the system applicability solution results are shown in FIG. 1. In the system applicability solution, the separation degree of the critical starting material of the litaxet from the adjacent impurity peaks is 4.5, and the retention time Rt is 20.747 minutes.
2.3 repeatability
Taking the key starting materials of the rituximab and the right amounts of the impurities A, B, C, D, E and F, precisely weighing, putting into a same measuring flask, adding a diluent to dissolve and dilute the materials to prepare a solution containing about 0.3mg of the compound of the formula I and about 0.3 mug of each impurity in 1ml of the solution as a standard test sample solution; precisely transferring 0.5ml of sample solution, placing into a 50ml measuring flask, diluting to scale with diluent, mixing, precisely transferring 1ml of the solution, placing into a 10ml measuring flask, and diluting with diluent to obtain about 0.3 μg of solution containing the compound of formula I per 1ml of solution as control solution. And precisely measuring the solution of the added standard sample and the control solution, sampling, and recording a chromatogram. And calculating the RSD of the contents of all related substances in 6 parts of the marked test sample solution according to the main component self-comparison method added with the correction factors. The RSD of the content of each detected relevant impurity in 6 parts of the labeled sample solution is less than 10.0 percent, which meets the requirements of high performance liquid chromatography on detection of the relevant substances.
2.4 linearity
And (3) taking a proper amount of the critical starting materials of the rituximab and the impurity reference substances thereof, adding a diluent for dissolving and diluting to prepare a solution with 3 mug of each 1ml of critical starting materials of the rituximab, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F serving as a linear stock solution. Respectively precisely transferring 1ml of stock solution into 20ml, 2ml into 25ml, 1ml into 10ml, 3ml into 25ml, 2ml into 10ml, 5ml into 10ml measuring flask, diluting with diluent to scale, and shaking; and taking the quantitative limiting solution as a linear test solution. The concentration was linearly regressed with peak area to obtain a linear equation (table 1), and the key starting materials of rituximab and its impurities had a good linear relationship in the linear range.
TABLE 1 Linear measurement results
2.5 detection limit and quantitative limit
And (3) preparing a series of solutions by taking proper amounts of key starting materials of the rituximab and impurity reference substances thereof, wherein the solution is used as a quantitative limiting solution when the S/N is more than or equal to 10, and the solution is used as a detection limiting solution when the S/N is more than or equal to 3. The quantitative limit and the detection limit of the critical starting materials and impurities of the rituximab are shown in Table 2.
TABLE 2 quantitative limit and detection limit results
2.6 accuracy
Taking a proper amount of each of the reference substances of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F, adding a diluent for dissolving and diluting to prepare a solution with 3 mug per 1ml, and taking the solution as an impurity mixed reference substance stock solution; taking appropriate amount of critical starting materials of Litasast, precisely measuring 0.2ml of impurity mixed reference substance stock solution, placing 10ml of the stock solution, 0.5ml of the stock solution, 10ml of the stock solution, 1ml of the stock solution, 10ml of the stock solution, 1.5ml of the stock solution, 3ml of the stock solution and a 10ml measuring flask respectively, diluting with diluent to scale, shaking, and collecting the sample solution with a recovery rate of 3 parts per concentration. The recovery rate and RSD of each impurity were calculated by the principal component self-comparison method with the correction factor added. The result shows that the recovery rate of each impurity at each concentration is between 80.0% and 120.0%, and the RSD is less than 10.0%, which meets the requirements of high performance liquid chromatography on detection of related substances.
Conclusion 3:
under the chromatographic condition, the key starting materials of the rituximab and impurities thereof can be completely separated, and the method has the advantages of strong specificity, good accuracy, high sensitivity, good repeatability and good system applicability, meets the technical requirements of drug quality research standards, and has stable and reliable results.
Comparative example 1:
1 chromatographic conditions:
chromatographic column: waters symmetry shield RP18 (4.6 mm. Times.50 mm,3.5 μm)
Supplementing column: welch Ghost Buster
Mobile phase a:10mmol/L disodium hydrogen phosphate solution (pH 5.0)
Mobile phase B: acetonitrile
Elution time: 60min
Column temperature: 35 DEG C
Detection wavelength: 220nm
Flow rate: 1.0ml/min
Sample injection amount: 20 μl of
A diluent: mobile phase A-mobile phase B (1:1)
2, the method comprises the following steps: the critical starting materials of the rituximab, the impurities A, B, C, D, E and F are precisely weighed and placed in the same measuring flask, and the solution containing about 0.2mg of the compound of the formula I and about 0.2 mug of each impurity is prepared as a system applicability solution by adding a diluent for dissolving and diluting the solution. 20 μl of the solution was precisely measured and injected into a liquid chromatograph, and the chromatogram was recorded, and the system applicability of the solution chromatograph is shown in FIG. 2.
Conclusion 3: under the chromatographic condition, the impurity C with retention time Rt of 0.583 min is too weak to be separated from the solvent peak, and the impurity peak in the blank solution is more, so that the impurity F (retention time Rt 27.098 min) is interfered, and the quantitative result and qualitative judgment of the impurity C and the impurity F are affected.
Comparative example 2:
1 chromatographic conditions:
chromatographic column: waters xselect HSS T3 (4.6 mm. Times.250 mm,5 μm)
Supplementing column: welch Ghost Buster
Mobile phase a:0.1% phosphoric acid water
Mobile phase B: acetonitrile
Elution time: 60min
Column temperature: 35 DEG C
Detection wavelength: 220nm
Flow rate: 1.0ml/min
Sample injection amount: 20 μl of
A diluent: 50% ACN
2, the method comprises the following steps: the critical starting materials of the rituximab, the impurities A, B, C, D, E and F are precisely weighed and placed in the same measuring flask, and the solution containing about 0.2mg of the compound of the formula I and about 5 mug of each impurity is prepared as a system applicability solution by adding a diluent for dissolving and diluting. 20 μl of the solution was precisely measured and injected into a liquid chromatograph, the chromatogram was recorded, and the system applicability of the solution chromatograph is shown in FIG. 3.
Conclusion 3: under the chromatographic condition, the peak shape of the impurity A with retention time Rt 29.827 min is poor, the peak separation degree of the impurity C with retention time Rt 3.748 min and the blank solvent is poor, and the blank solvent can interfere with the impurity C, so that the quantitative result and the qualitative judgment of the impurity C are affected. The blank has obvious bulge, and influences the impurity detection sensitivity.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (7)
1. A method for detecting critical starting materials of ritalski comprises the steps of adopting HPLC (high performance liquid chromatography) to separate and detect critical starting materials of ritalski and related impurities thereof, adopting a chromatographic column which takes octadecylsilane chemically bonded silica as a filler, adopting a mobile phase A and a mobile phase B to carry out gradient elution, and entering a detector to carry out detection; the related impurities comprise one or more of impurities A, B, C, D, E and F, and the specific structural formula is shown as follows:
The mobile phase A is perchloric acid aqueous solution, the mobile phase B is organic solvent,
the key starting material of the rituximab is 2- (tert-butoxycarbonyl) -5, 7-dichloro-1, 2,3, 4-tetrahydroisoquinoline-6-carboxylic acid, which is the key starting material for synthesizing the rituximab and has a chemical formula of C 15 H 17 Cl 2 NO 4 The structural formula is shown as the following formula (I), and the invention is also called as a compound of the formula I or a key starting material of the litazost:
the gradient elution settings were as follows:
the flow rate of the mobile phase for elution is 0.8-1.2ml/min.
2. The method according to claim 1, wherein the mobile phase A is an aqueous perchloric acid solution, the ratio of perchloric acid in the aqueous perchloric acid solution is 0.1% -0.2%, and the mobile phase B is acetonitrile.
3. The method according to claim 2, wherein the ratio of perchloric acid in the aqueous perchloric acid solution is 0.15%.
4. The method of claim 1, wherein the gradient elution is set as follows:
5. the method according to claim 1, wherein the filler particles of octadecylsilane chemically bonded silica have a particle size of 2 to 5 μm; the column temperature of the chromatographic column is 25-35 ℃.
6. The method of claim 1, wherein the detector has a detection wavelength of 220±5nm.
7. The method according to claim 1, wherein the related impurities are impurities a, B, C, D, E, F controls, comprising the steps of:
1) Preparing a test sample solution: taking a proper amount of critical starting materials of the ritodynamic, precisely weighing, adding 2/5 volume of methanol for ultrasonic dissolution, and diluting with water to prepare a solution containing about 0.3mg per 1 ml;
2) Preparing a control solution: taking a proper amount of test solution, and diluting with a diluent to prepare a solution with about 0.3 mug in each 1ml serving as a control solution;
3) Preparing a system applicability solution: taking a critical starting material of rituximab and impurities A, B, C, D, E and F thereof, and dissolving and diluting the critical starting material with a diluent to prepare a system applicability solution;
4) Taking the solution with the suitability of the system in the step 3), carrying out high performance liquid chromatography analysis, recording a chromatogram, determining the retention time of the critical starting materials of the litaxest and the impurities thereof, respectively taking the sample solution in the step 1) and the control solution in the step 2), carrying out sample injection, calculating the content of the impurities in the critical starting materials of the litaxest in the sample solution according to a main component self-control method,
the diluent is a methanol-water (volume ratio of 40:60) solution.
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