CN113820413B - Method for separating and measuring tricitabine phosphate and impurities by high performance liquid chromatography - Google Patents
Method for separating and measuring tricitabine phosphate and impurities by high performance liquid chromatography Download PDFInfo
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- G—PHYSICS
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- 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
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- 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
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Abstract
The invention relates to the field of analytical chemistry, and particularly discloses a method for separating and determining tricitabine phosphate and impurities by using a high performance liquid chromatography. The invention improves the relevant chromatographic condition parameters, so that the tricitabine phosphate and the impurities can be better separated, and the method is utilized to carry out qualitative and quantitative analysis on the main drug and the impurities thereof. The method has good specificity, is not interfered by blank solvents and other unknown impurities, has the separation degree between the main peak and the impurity B of 3.99 and the separation degrees between other known impurities of more than 1.5, and has the advantages of simplicity, rapidness, high accuracy and the like.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for separating and determining trictinib phosphate and impurities by using a high performance liquid chromatography.
Background
The incarnib phosphate is a JAK1/JAK2 inhibitor, is mainly used for middle-risk or high-risk adult patients with primary myelofibrosis (also called chronic idiopathic myelofibrosis), myelofibrosis secondary to polycythemia vera or myelofibrosis secondary to primary thrombocythemia, and treats disease-related splenomegaly or disease-related symptoms.
The dose of the tricitabine phosphate needs to be strictly controlled in the treatment process, the platelet content in a patient body is periodically monitored, and a doctor adjusts the dose according to related indexes. However, in the production process, a lot of impurities are generated, and the impurities tend to influence the content of the active ingredients of the main drug, so that the detection of the impurities in the production process is particularly important.
High performance liquid chromatography is often applied to detection of various drug impurities, but the chromatographic conditions of related substances of the imported registration standard (JX 20140057) of the reeklatonia phosphate tablet are not suitable for detection of finished drugs of all manufacturers, and no specific method capable of simultaneously separating and determining the main drug and the related impurities exists at present, which has adverse effects on the production efficiency and the finished product qualification rate of a drug enterprise.
Disclosure of Invention
In order to solve the problems, the invention provides a method for separating and determining the lucigenin phosphate and the impurities by using a high performance liquid chromatography, the invention searches for chromatographic conditions under which the lucigenin phosphate and the impurities can be well separated, and the method is used for carrying out qualitative and quantitative analysis on the main drug and the impurities thereof.
One of the purposes of the invention is to provide a method for separating the tricuspid phosphate and impurities by using a high performance liquid chromatography, and the specific technical scheme is as follows:
the mobile phase A is potassium dihydrogen phosphate, and the mobile phase B is methanol-acetonitrile; the chromatographic column filler is octadecylsilane chemically bonded silica; the impurities include:
specifically, the impurities further include:
specifically, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile is 50:50.
specifically, the elution ratio is:
0min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
2min, wherein the volume ratio of the mobile phase A to the mobile phase B is 70:30,
22min, the volume ratio of the mobile phase A to the mobile phase B is 48:52,
27min, the volume ratio of the mobile phase A to the mobile phase B is 20:80,
35min, the volume ratio of the mobile phase A to the mobile phase B is 20:80,
36min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
45min, the volume ratio of the mobile phase A to the mobile phase B is 70:30.
specifically, the pH value of the mobile phase A is 3.5 +/-0.1.
Specifically, the flow rate was 1. + -. 0.1ml/min.
Specifically, the concentration of the potassium dihydrogen phosphate is 0.03 +/-0.005 mol/L.
Specifically, the detection wavelength is 220 +/-10 nm.
Specifically, the column temperature was 35. + -. 2 ℃.
Specifically, the diluent is methanol.
The invention also aims to provide a method for determining the contents of the tricitabine phosphate and impurities by using the high performance liquid chromatography, and the specific technical scheme is as follows:
measuring the content of the tricitabine phosphate or impurities by adopting an internal standard method, wherein a mobile phase A is monopotassium phosphate, and a mobile phase B is methanol-acetonitrile; the chromatographic column filler is octadecylsilane chemically bonded silica; the elution ratio was: the impurities include:
/>
specifically, the impurities further include:
specifically, the elution ratio is:
0min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
2min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
22min, the volume ratio of the mobile phase A to the mobile phase B is 48:52,
27min, the volume ratio of the mobile phase A to the mobile phase B is 20:80,
35min, the volume ratio of the mobile phase A to the mobile phase B is 20:80,
36min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
45min, the volume ratio of the mobile phase A to the mobile phase B is 70:30.
specifically, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile is 50:50.
specifically, the pH value of the mobile phase A is 3.5 +/-0.1.
Specifically, the flow rate was 1. + -. 0.1ml/min.
Specifically, the concentration of the potassium dihydrogen phosphate is 0.03 +/-0.005 mol/L.
Specifically, the detection wavelength is 220 +/-10 nm.
Specifically, the column temperature was 35. + -. 2 ℃.
Specifically, the diluent is methanol.
Specifically, the concentration of the control solution is 2 + -0.05 μ g/ml, and the concentration of the test solution is 2.0 + -0.05 mg/ml.
The invention has the advantages that: the method has strong specificity, can effectively separate all known impurities and unknown impurities, effectively control the quality of the finished product of the reed cotinib phosphate, can realize the effective separation of 8 impurities in the finished product, namely the impurity B, the impurity C, the impurity D, the impurity E, the impurity F, the impurity G, the impurity H and the impurity I, in a set of HPLC method, has good specificity, is not interfered by a blank solvent and other unknown impurities, has the separation degree between a main peak and the impurity B of 3.99 which is more than 1.5, has the separation degree between other known impurities of more than 1.5, meets the requirements of related substances, and has the advantages of simplicity, rapidness, high accuracy and the like.
Drawings
FIG. 1 is a white solvent HPLC chart in example 2;
FIG. 2 is a HPLC chart of the mixed solution in example 2;
FIG. 3 is a HPLC chart of a localized solution of impurity G in example 2;
FIG. 4 is a HPLC chart of a localized solution of impurity B in example 2;
FIG. 5 is a HPLC chart of a localized solution of impurity C in example 2;
FIG. 6 is a HPLC chart of a localized solution of impurity D in example 2;
FIG. 7 is a HPLC chart of a localized solution of impurity E in example 2;
FIG. 8 is a HPLC chart of a localized solution of impurity F in example 2;
FIG. 9 is a HPLC chart of a localized solution of impurity H in example 2;
FIG. 10 is a HPLC chart of a localized solution of impurity I in example 2;
FIG. 11 is a HPLC chart of the test solution in example 2;
FIG. 12 is an HPLC plot of elution gradient ratio 1 in example 3;
FIG. 13 is an HPLC chart of elution gradient ratio 2 in example 3;
FIG. 14 is an HPLC plot of elution gradient ratio 3 in example 3;
FIG. 15 is an HPLC plot of elution gradient ratio 4 in example 3;
FIG. 16 is an HPLC plot of elution gradient ratio 5 in example 3;
FIG. 17 is an HPLC plot of elution gradient ratio 6 in example 3;
FIG. 18 is an HPLC chart of mixed solution 1 in example 4;
FIG. 19 is an HPLC chart of mixed solution 2 in example 4;
FIG. 20 is an HPLC chart of mixed solution 3 in example 4;
FIG. 21 is an HPLC chart of the mixed solution 4 in example 4;
FIG. 22 is an HPLC chart of the mixed solution 5 in example 4;
FIG. 23 is an HPLC chart of the mixed solution 6 in example 4;
FIG. 24 is an HPLC chart of the mixed solution 7 in example 4;
FIG. 25 is an HPLC chart of mixed solution 8 in example 4.
Detailed Description
The present invention is further described in detail by the following examples, which should be understood that the present invention is not limited to the particular examples described herein, but is intended to cover modifications within the spirit and scope of the present invention.
The invention mainly separates and measures the compounds shown in the table 1, and searches related elution condition parameters of the high performance liquid chromatography, so that the main medicine has better separation degree with related impurities, thereby accurately measuring the content of each substance.
TABLE 1 separation of assay substances
Example 1
The embodiment specifically describes a method for separating the tricitabine phosphate and impurities by using a high performance liquid chromatography, and the specific technical scheme is as follows: the mobile phase A is potassium dihydrogen phosphate, and the mobile phase B is methanol-acetonitrile; the chromatographic column filler is octadecylsilane chemically bonded silica;
the impurities include:
the method also enables the following impurities to be separated:
specifically, the elution ratio is:
0min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
2min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
22min, the volume ratio of the mobile phase A to the mobile phase B is 48:52,
27min, the volume ratio of the mobile phase A to the mobile phase B is 20:80,
35min, the volume ratio of the mobile phase A to the mobile phase B is 20:80,
36min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
45min, the volume ratio of the mobile phase A to the mobile phase B is 70:30.
specifically, the volume ratio of methanol to acetonitrile in methanol-acetonitrile is 50:50.
specifically, the pH value of the mobile phase A is 3.5 +/-0.1.
Specifically, the flow rate was 1. + -. 0.1ml/min.
Specifically, the concentration of the potassium dihydrogen phosphate is 0.03 +/-0.005 mol/L.
Specifically, the detection wavelength is 220 +/-10 nm.
Specifically, the column temperature was 35. + -. 2 ℃.
Specifically, the diluent is methanol.
The method described in this embodiment can not only separate the ruckutinib phosphate and the impurities, but also measure the content of each substance according to the conventional methods, including but not limited to the external standard method and the internal standard method, and this embodiment only describes the optimal method for separating and/or determining the ruckutinib phosphate and the impurities, and the relevant parameter conditions, including the elution gradient ratio, the kind and concentration of the buffer salt, the flow rate, the column temperature, etc., can be adjusted within a certain range, i.e. the claimed scope of the present invention is not limited to the scope covered by embodiment 1, for example, the following embodiment 3 describes other feasible condition parameters of the high performance liquid chromatography.
Example 2
This example further illustrates a method for determining the content of tricitabine phosphate and impurities by high performance liquid chromatography on the basis of example 1, that is, relevant condition parameters are specifically defined as follows:
1. parameters of elution conditions
In the present example, high performance liquid chromatography is used to measure the content of trictinib phosphate and impurities, and the specific experimental conditions and parameters are shown in table 2.
TABLE 2 separation and determination of the conditional parameters of the triceratinib phosphate and the impurities by HPLC
2. Solution preparation
Impurity B stock solution: accurately weighing 25.56mg of the impurity B reference substance, placing the impurity B reference substance in a 25ml measuring flask, adding acetonitrile to dissolve and dilute the impurity B reference substance to a scale, and shaking up to obtain the product.
Impurity C stock solution: accurately weighing 10.46mg of impurity C reference substance, placing into a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up to obtain the final product.
Impurity D stock solution: accurately weighing 24.69mg of the impurity D reference substance, placing the impurity D reference substance into a 25ml measuring flask, adding a diluent to dissolve and dilute the impurity D reference substance to a scale, and shaking up to obtain the impurity D reference substance.
Impurity E stock solution: accurately weighing 25.04mg of the impurity E reference substance, placing in a 25ml measuring flask, adding a diluent to dissolve and dilute to scale, and shaking up to obtain the final product.
Impurity F stock solution: accurately weighing 25.67mg of impurity F reference substance, placing into a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up to obtain the final product.
Impurity G stock solution: accurately weighing 8.10mg of impurity G as a reference substance, placing in a 25ml measuring flask, adding a diluent to dissolve and dilute to scale, shaking up, filtering, and taking a subsequent filtrate to obtain the final product.
Impurity H (D-DMTA) stock solution: accurately weighing 25.02mg of impurity H reference substance, placing in a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up to obtain the final product.
Impurity I (p-methoxybenzoic acid) stock solution: precisely weighing 25.32mg of p-anisic acid reference substance, placing in a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up to obtain the final product.
The HPLC profile of the blank solvent is shown in FIG. 1.
Impurity mixed stock solution: precisely transferring 3.0ml of impurity B, impurity D, impurity E, impurity F, impurity H, impurity I stock solution and 7.5ml of impurity C stock solution into the same 100ml measuring flask, adding acetonitrile to dilute to scale, and shaking up to obtain the final product.
Mixing the solution: precisely weighing 49.98mg of a sample, placing in a 25ml measuring flask, precisely weighing 2.5ml of impurity mixed stock solution and 5ml of impurity G positioning solution, adding diluent to dissolve and dilute to scale, and shaking up to obtain the final product, wherein HPLC chart is shown in FIG. 2.
Each impurity localization solution: precisely transferring 0.25ml of impurity C stock solution and 0.1ml of other impurity stock solutions, respectively placing into different 10ml measuring bottles, adding diluent to dilute to scale, and shaking to obtain the final product, wherein HPLC chart is shown in FIGS. 3-10.
Test solution: precisely weighing 50.03mg of the test sample, placing the test sample in a 25ml measuring flask, adding a diluent to dissolve and dilute the test sample to a scale, and shaking up to obtain the HPLC chart shown in figure 11.
3. Results of the experiment
The experimental results shown in table 3 can be obtained by combining fig. 1-11, wherein the blank solvent does not interfere the measurement of related substances of the product, the separation degrees between known impurity peaks, main peaks and adjacent impurity peaks are all larger than 1.5, the measurement is not interfered, and the separation degree is good.
TABLE 3 results of the substance separation experiment
Example 3
Other feasible chromatographic conditions are described in this example, including elution gradient ratios, buffer salt species and concentrations, flow rates, column temperatures, etc.
1. Investigation of different elution gradient ratios
A chromatographic column: agilent ZORBAX SB-C18.6 mm X250mm, 5 μm
Column temperature: 25 deg.C
Flow rate: 1.0ml/min
Mobile phase A:0.03mol/L potassium dihydrogen phosphate aqueous solution (pH adjusted to 3.5 with phosphoric acid)
Mobile phase B: methanol
Mobile phase C: acetonitrile
Elution gradient ratio 1: (HPLC chart is shown in FIG. 12, the separation degree of each impurity meets the requirement)
Elution gradient ratio 2: (HPLC chart is shown in FIG. 13, the separation degree of each impurity meets the requirement)
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Mobile phase C (%) |
| 0 | 70 | 15 | 15 |
| 2 | 70 | 15 | 15 |
| 20 | 60 | 20 | 20 |
| 25 | 20 | 40 | 40 |
| 40 | 20 | 40 | 40 |
| 41 | 70 | 15 | 15 |
| 50 | 70 | 15 | 15 |
Elution gradient ratio 3: (HPLC chart is shown in FIG. 14, the separation degree of each impurity meets the requirement)
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Mobile phase C (%) |
| 0 | 75 | 12.5 | 12.5 |
| 2 | 75 | 12.5 | 12.5 |
| 22 | 48 | 26 | 26 |
| 27 | 20 | 40 | 40 |
| 45 | 20 | 40 | 40 |
| 46 | 75 | 12.5 | 12.5 |
| 55 | 75 | 12.5 | 12.5 |
Elution gradient ratio 4: (HPLC chart is shown in FIG. 15, the separation degree of each impurity meets the requirement)
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Mobile phase C (%) |
| 0 | 70 | 15 | 15 |
| 35 | 20 | 40 | 40 |
| 45 | 20 | 40 | 40 |
| 46 | 70 | 15 | 15 |
| 55 | 70 | 15 | 15 |
Elution gradient ratio 5: (HPLC chart is shown in FIG. 16, separation degree of each impurity meets the requirement)
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Mobile phase C (%) |
| 0 | 70 | 15 | 15 |
| 40 | 20 | 40 | 40 |
| 50 | 20 | 40 | 40 |
| 51 | 70 | 15 | 15 |
| 60 | 70 | 15 | 15 |
Elution gradient ratio 6: (HPLC chart is shown in FIG. 17, the separation degree of each impurity meets the requirement)
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Mobile phase C (%) |
| 0 | 70 | 15 | 15 |
| 45 | 20 | 40 | 40 |
| 55 | 20 | 40 | 40 |
| 56 | 70 | 15 | 15 |
| 65 | 70 | 15 | 15 |
2. Buffer salt species and concentration
(1) Mobile phase A:0.03mol/L ammonium dihydrogen phosphate solution (pH 3.5 adjusted with phosphoric acid), and the rest of the chromatographic conditions were the same as in example 2, with the respective degrees of separation of impurities being satisfactory.
(2) Mobile phase A:0.03mol/L sodium dihydrogen phosphate aqueous solution (pH was adjusted to 3.5 with phosphoric acid), and the remaining chromatographic conditions were the same as in example 1, and the degree of separation of each impurity was satisfactory.
(3) Mobile phase A:0.02mol/L and 0.04mol/L potassium dihydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid), and the rest of the chromatographic conditions were the same as in example 2, and the separation degree of each impurity was satisfactory. When the concentration is 0.01mol/L, the remaining chromatographic conditions are the same as in example 2, and the degree of separation is unsatisfactory.
3. Influence of flow velocity on impurity separation
The flow rates were 0.8ml/min and 1.2ml/min, the chromatographic conditions were the same as in example 2, and the degrees of separation of the impurities were satisfactory.
4. Column temperature
The column temperature was 25 ℃,35 ℃ and 40 ℃, the other chromatographic conditions were the same as in example 2, and the separation degree of each impurity was satisfactory.
Example 4
In the embodiment, relevant important parameters of the previous experimental method are relatively searched, wherein the relevant important parameters comprise the improvement of the type, the pH value, the ratio and the elution gradient of the mobile phase, and the innovation of the method is highlighted.
1. The existing standard chromatographic condition detection can not separate main drug and related impurities recorded in the main drug
Earlier it was found that the use of the tablet of reecotinib phosphate was subject to the import registration standard (JX 8978 zxft) 8978) the chromatographic conditions of the relevant substances are not effective in separating the principal drugs and the relevant impurities. As shown in fig. 18, which is an HPLC chart of the orcocotinib phosphate tablet obtained by using chromatographic conditions (shown in table 4) of related substances of the imported registered standard (JX 20140057). Therefore, the separation results of the related substance method on the mixed solution of the known impurities and the main component show that: the impurity C is not separated from the impurity F, the impurity B is not separated from the main peak, and each impurity peak is trailing, so that the method is not applicable, and the chromatographic conditions of related substances in the imported registration standard (JX 20140057) of the reed canatinib phosphate tablet are not suitable for detecting related impurities in the finished product of me.
TABLE 4 Luketinib phosphate tablets Using chromatographic conditions for related substances according to the import registration Standard (JX 20140057)
2. Search for mobile phase meeting standard
When water is used as the mobile phase A and methanol is used as the mobile phase B, specific chromatographic conditions and elution modes are shown in Table 5, HPLC is shown in FIG. 19, and a plurality of impurities cannot be effectively separated. Under the chromatographic conditions, the impurity H and the impurity C are not separated, and the peak is earlier and is not retained; the p-methoxybenzoic acid is coincided with the impurity F; impurity E and impurity G do not reach baseline separation, so the chromatographic condition is not suitable for detecting related impurities of the product.
TABLE 5 search for mobile phase
3. Influence of mobile phase pH value on impurity separation condition
TABLE 6 influence of mobile phase pH on impurity separation
4. Influence of mobile phase ratio on impurity separation
TABLE 7 influence of the mobile phase ratio on the separation of impurities
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.
Claims (2)
1. The method for determining the tricitabine phosphate and the impurities by the high performance liquid chromatography is characterized by comprising the following steps: octadecylsilane chemically bonded silica is used as a chromatographic column filler, dihydric phosphate with the concentration of 0.03 +/-0.005 mol/L is used as a mobile phase A, methanol-acetonitrile is used as a mobile phase B for gradient elution, and the flow rate is 1 +/-0.1 ml/min; the detection wavelength is 220 +/-10 nm; the specification of the chromatographic column is 4.6mm multiplied by 250mm and 5 mu m; the pH value of the mobile phase A is 3.5 +/-0.1, and the pH value is adjusted by phosphoric acid;
the volume ratio of methanol to acetonitrile in the methanol-acetonitrile is 50:50;
the elution ratio was:
0min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
2min, wherein the volume ratio of the mobile phase A to the mobile phase B is 70:30,
22min, and the volume ratio of the mobile phase A to the mobile phase B is 48:52,
27min, the volume ratio of the mobile phase A to the mobile phase B is 20:80,
35min, the volume ratio of the mobile phase A to the mobile phase B is 20:80,
36min, the volume ratio of the mobile phase A to the mobile phase B is 70:30,
45min, the volume ratio of the mobile phase A to the mobile phase B is 70:30, of a nitrogen-containing gas;
the impurities include:
2. the method of claim 1, wherein the sample solution is prepared by: precisely weighing the sample, dissolving in methanol, diluting, and shaking.
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| WO2016035014A1 (en) * | 2014-09-01 | 2016-03-10 | Sun Pharmaceutical Industries Limited | Processes for the preparation of ruxolitinib phosphate |
| CN110940742A (en) * | 2019-09-18 | 2020-03-31 | 北京陆道培生物技术有限公司 | Method for detecting concentration of blood disease related medicine and application |
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| US20150197525A1 (en) * | 2012-06-15 | 2015-07-16 | Concert Pharmaceuticals, Inc. | Deuterated derivatives of ruxolitinib |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016035014A1 (en) * | 2014-09-01 | 2016-03-10 | Sun Pharmaceutical Industries Limited | Processes for the preparation of ruxolitinib phosphate |
| CN110940742A (en) * | 2019-09-18 | 2020-03-31 | 北京陆道培生物技术有限公司 | Method for detecting concentration of blood disease related medicine and application |
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| Biswal Sabyasachi 等.A new stability indicating high performance liquid chromatography method for the estimation of ruxolitinib in bulk and tablet dosage form.Pharmaceutical Methods.2019,第10卷(第10期),第53-57页. * |
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