CN111257491A - HPLC method for detecting cyclopropylamine in Levatinib mesylate - Google Patents
HPLC method for detecting cyclopropylamine in Levatinib mesylate Download PDFInfo
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- CN111257491A CN111257491A CN201811458909.6A CN201811458909A CN111257491A CN 111257491 A CN111257491 A CN 111257491A CN 201811458909 A CN201811458909 A CN 201811458909A CN 111257491 A CN111257491 A CN 111257491A
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- cyclopropylamine
- derivatization
- acetonitrile
- solvent
- buffer solution
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- HTJDQJBWANPRPF-UHFFFAOYSA-N Cyclopropylamine Chemical compound NC1CC1 HTJDQJBWANPRPF-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 12
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 title abstract description 22
- 238000001212 derivatisation Methods 0.000 claims abstract description 64
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 20
- 239000007853 buffer solution Substances 0.000 claims abstract description 11
- 239000003960 organic solvent Substances 0.000 claims abstract description 10
- 150000003839 salts Chemical class 0.000 claims abstract description 9
- 150000001412 amines Chemical class 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 66
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 52
- 239000002904 solvent Substances 0.000 claims description 49
- 239000000243 solution Substances 0.000 claims description 41
- 238000001514 detection method Methods 0.000 claims description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- VYZAHLCBVHPDDF-UHFFFAOYSA-N Dinitrochlorobenzene Chemical compound [O-][N+](=O)C1=CC=C(Cl)C([N+]([O-])=O)=C1 VYZAHLCBVHPDDF-UHFFFAOYSA-N 0.000 claims description 26
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 229960001429 lenvatinib mesylate Drugs 0.000 claims description 4
- HWLFIUUAYLEFCT-UHFFFAOYSA-N lenvatinib mesylate Chemical compound CS(O)(=O)=O.C=12C=C(C(N)=O)C(OC)=CC2=NC=CC=1OC(C=C1Cl)=CC=C1NC(=O)NC1CC1 HWLFIUUAYLEFCT-UHFFFAOYSA-N 0.000 claims description 4
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 claims description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims description 2
- 239000008351 acetate buffer Substances 0.000 claims description 2
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 2
- -1 p-nitrophenyl N-succinimidyl acetate Chemical compound 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 37
- 239000012535 impurity Substances 0.000 description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 18
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 18
- 239000005695 Ammonium acetate Substances 0.000 description 18
- 229940043376 ammonium acetate Drugs 0.000 description 18
- 235000019257 ammonium acetate Nutrition 0.000 description 18
- 238000007865 diluting Methods 0.000 description 17
- 238000005303 weighing Methods 0.000 description 15
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 14
- 239000013558 reference substance Substances 0.000 description 12
- 239000007864 aqueous solution Substances 0.000 description 11
- 238000001816 cooling Methods 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 238000007789 sealing Methods 0.000 description 11
- 238000005406 washing Methods 0.000 description 10
- 229960000583 acetic acid Drugs 0.000 description 9
- 239000012362 glacial acetic acid Substances 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 238000000926 separation method Methods 0.000 description 8
- 238000002156 mixing Methods 0.000 description 7
- 239000012488 sample solution Substances 0.000 description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000012088 reference solution Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- YBADLXQNJCMBKR-UHFFFAOYSA-M (4-nitrophenyl)acetate Chemical compound [O-]C(=O)CC1=CC=C([N+]([O-])=O)C=C1 YBADLXQNJCMBKR-UHFFFAOYSA-M 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- ZSPLOATUDIRNAS-PPHPATTJSA-N levofloxacin mesylate Chemical compound CS(O)(=O)=O.C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 ZSPLOATUDIRNAS-PPHPATTJSA-N 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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
- G01N30/89—Inverse chromatography
Abstract
The invention discloses an HPLC method for detecting cyclopropylamine in lervatinib mesylate, which adopts a reversed phase chromatographic column, uses a derivatization reagent to perform derivatization treatment on a sample to be detected, and a mobile phase is selected from a salt buffer solution containing an amine substance and an organic solvent. The method provided by the invention can accurately detect the content of cyclopropylamine in the lervatinib mesylate, is simple and easy to implement, has low analysis cost and accurate and reliable result, and is convenient for controlling the product quality in the production and quality control processes.
Description
Technical Field
The invention relates to a HPLC analysis method of cyclopropylamine.
Background
Cyclopropylamine (SCR-4341) is the starting material used in the third step of the synthesis of lenvatinib mesylate. The cyclopropylamine is free of ultraviolet absorption, a gas-phase headspace method is usually adopted for measuring the cyclopropylamine, but the lervatinib mesylate is easily degraded to generate the cyclopropylamine under a high-temperature condition to interfere the detection, and in addition, a CAD detector is adopted for measuring, so that the detection sensitivity is low, and the content of the cyclopropylamine in the lervatinib mesylate cannot be well controlled.
The prior art does not record a specific analysis method for detecting cyclopropylamine from Levatinib mesylate, and the HPLC pre-column derivatization method is adopted to determine the cyclopropylamine, so that the interference detection of the cyclopropylamine generated by high-temperature degradation of the Levatinib mesylate can be avoided, and meanwhile, the detection method has the advantages of good separation degree, single product after derivatization, no blank interference peak and capability of accurately detecting the content of the cyclopropylamine in a sample.
Disclosure of Invention
The invention provides an HPLC method for detecting cyclopropylamine in lervatinib mesylate, which is characterized in that: adopting a reversed phase chromatographic column; performing derivatization treatment on a sample to be detected by using a derivatization reagent; the mobile phase is selected from salt buffer solution containing amine substances and organic solvent, and the detection wavelength is 215-356nm, preferably 356 nm.
The derivatization reagent is selected from 2, 4-dinitrochlorobenzene, 2, 4-dinitrofluorobenzene, benzoyl chloride or N-succinimide p-nitrophenylacetate.
The derivatization treatment is to use a derivatization reagent to react with cyclopropylamine to generate cyclopropylamine derivatives with ultraviolet absorption, preferably
Is selected as
Sodium carbonate is added in the derivatization treatment process, and the mass ratio of the sodium carbonate to a test sample is 40: 1-60: 1, preferably 50: 1.
Furthermore, the derivatization time is 1.5-12 h.
Further, the derivatization temperature is 60-80 ℃, and preferably 75 ℃.
The salt buffer solution is acetate buffer solution and formate buffer solution.
The pH value of the salt buffer solution containing the amine substances is 4.0-6.0, and preferably 5.0.
Furthermore, the volume ratio of the salt buffer solution containing the amine substance to the organic solvent is 1: 1-1: 6, preferably 1: 1.2-1: 4, more preferably 9:11, and preferably the volume of the organic solvent is not changed or increased during the elution process.
The organic solvent is selected from one or more of acetonitrile, methanol and ethanol.
The organic solvent is preferably acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol is 2: 1-1: 3, preferably 1:1. .
Further, the method comprises the step of dissolving a sample to be detected by using a solvent, wherein the solvent is composed of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 2: 1-6: 1, preferably 4: 1.
The derivatization in the present invention is a chemical transformation of a compound into a substance of similar chemical structure.
Advantageous effects
1. The method provided by the invention can accurately detect the content of cyclopropylamine in the lervatinib mesylate, and avoids the problem that the content of cyclopropylamine cannot be accurately determined due to decomposition of the lervatinib mesylate caused by high temperature when a gas-phase headspace method is used for detection.
2. The method provided by the invention can separate the cyclopropylamine from other impurities, and the separation degrees are all more than 1.5 and meet the standard.
3. The method provided by the invention can accurately detect the content of cyclopropylamine in the lervatinib mesylate, has good durability of derivatization conditions and single product, does not interfere with the detection of other impurities, is simple and easy to implement, has low analysis cost and accurate and reliable result, and is convenient for the control of product quality in the production and quality control processes.
Drawings
FIG. 1: EXAMPLE 1 Cyclopropylamine derivatized chromatographic Peak Profile
FIG. 2: example 2 detection System suitability map
FIG. 3: HPLC detection profile of control solution in example 5
FIG. 4: HPLC detection profile of test solution in example 5
FIG. 5: HPLC detection Profile in example 11
FIG. 6: HPLC detection Profile in example 12
Detailed Description
The present invention is further illustrated by the following examples, it being understood that these examples are given by way of illustration only and not by way of limitation, and that the invention is accordingly to be construed as limited only by the process claimed herein.
The cyclopropylamine (SCR-4341) used in the detection in the examples below was supplied by Annagi chemical reagent and samples of lenvatinib mesylate were supplied by Jiangsu Protoyor pharmaceutical, under lots 150115, 150119, 150125.
Example 1:
taking a proper amount of cyclopropylamine, adding a proper amount of a solvent [ acetonitrile-water (80:20) ], adding a derivatization reagent 2, 4-dinitrochlorobenzene solution to prepare a control solution, and carrying out full-wavelength spectrum acquisition by using a Diode Array Detector (DAD), wherein the result shows that the maximum absorption of SCR-4341 after derivatization chromatographic peaks is 215nm, 264nm and 356nm, the detection sensitivity is higher and the interference is smaller under the wavelength of 356nm, so that the wavelength of 356nm is selected as the measurement wavelength of the product. The detection spectrum is shown in figure 1.
Example 2
1) Chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding 1ml glacial acetic acid, mixing, adjusting pH to 5.0) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
(1) Precisely weighing a proper amount of SCR-4341 reference substances to prepare a solution containing 7.5mg of SCR-4341 per 1ml, precisely weighing 2ml to 20ml of headspace bottles, adding 10mg of levofloxacin mesilate, precisely weighing 0.5ml of sodium carbonate aqueous solution (obtained by taking 2.5g of sodium carbonate and adding 25ml of water for dissolution), adding 0.2ml of derivatization reagent 2, 4-dinitrochlorobenzene solution (obtained by taking 100mg to 20ml of 2, 4-dinitrochlorobenzene measuring bottles, adding acetonitrile for dilution to scale and shaking up), sealing, pressing a cover, carrying out water bath at 75 ℃ for 5 hours, cooling, washing with a solvent for multiple times, completely transferring to 10ml measuring bottles, adding the solvent for dilution to scale, shaking up, and preparing a system applicability test solution.
(2) Precisely weighing a proper amount of each of the impurities SCR-4338, SCR-4452, SCR-4453 and SCR-4342 to prepare a solution containing about 7.5mg of each impurity per 1ml, precisely weighing 2ml to 20ml of a headspace bottle, adding 10mg of mevalontinib mesylate, precisely weighing 0.5ml of a sodium carbonate aqueous solution (2.5 g of sodium carbonate is taken and 25ml of water is added for dissolving), adding a derivatization reagent 2, 4-dinitrochlorobenzene solution (100 mg to 20ml of 2, 4-dinitrochlorobenzene is taken, acetonitrile is added for diluting to a scale and shaking uniformly) 0.2ml, sealing, capping, carrying out water bath at 75 ℃ for 5 hours, cooling, washing with a solvent for multiple times, completely transferring to a 10ml measuring bottle, and diluting the solvent to the scale to prepare a reference solution containing the amino impurities.
TABLE 1 comparison of impurities
3) And (3) measuring results:
the retention time of the derivatized product of cyclopropylamine was 5.041 minutes. No other chromatographic peak exists in the chromatogram of the system applicability test solution in the vicinity of 5 minutes; no obvious chromatographic peak is observed in the chromatogram of the amino impurity control solution in the vicinity of 5 minutes, so that the existence of other impurities does not interfere with the detection of the derivative product of the cyclopropylamine. Meanwhile, acetonitrile-water (80:20) is selected as a solvent, the derivatization component and the sample of the impurity cyclopropylamine have better solubility, the derivatization peak type of the impurity cyclopropylamine is good, and a blank solvent does not interfere with detection. The detection result is shown in FIG. 2
Example 3:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding 1ml glacial acetic acid, mixing, adjusting pH to 5.0) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Precisely weighing a proper amount of a cyclopropylamine reference substance, adding a solvent acetonitrile-water (80:20) to quantitatively dilute the cyclopropylamine reference substance to prepare a solution containing about 7 mu g of cyclopropylamine per 1ml of the solution as a reference substance solution, taking 2ml of a reference substance stock solution, placing the reference substance stock solution in a 20ml headspace bottle, adding 0.1ml or 1ml of a derivatization reagent 2, 4-dinitrochlorobenzene solution (1mg/ml), and respectively examining derivatization at 40 ℃, 60 ℃ and 80 ℃ for 1 hour, wherein the results show that the peak of the cyclopropylamine derivative in the sample solution is very small (the peak area is less than 2) under the conditions of 40 ℃ and 60 ℃, and the detection sensitivity is low. Therefore, 80 ℃ is adopted as the derivatization temperature, and the addition amount and the derivatization time of different derivatization reagents are simultaneously examined.
3) And (3) measuring results:
the results in the table show that the derivatization temperature is 80 ℃, the time is 5 hours, the derivatization condition is stable, and the derivatization time length is moderate. Because the derivatization temperature is 80 ℃ close to the boiling point of acetonitrile, the volatilization amount of the acetonitrile in the solvent is large, a test product can be separated out from the solution, and the repeatability of the derivatization reaction is influenced. The derivatization temperature is adjusted to be 75 ℃, the derivatization time is respectively examined for 4.5 hours, 5 hours and 5.5 hours, and the results of three samples have good repeatability.
TABLE 1 Cyclopropylamine derivatization durability test results
Example 4:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding 1ml glacial acetic acid, mixing, adjusting pH to 5.0) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Precisely weighing 10mg of a sample to be tested, placing the sample into a 20ml headspace bottle, adding 2ml of a solvent, sequentially precisely weighing 0.5ml of a sodium carbonate aqueous solution (2.5 g of sodium carbonate is taken, 25ml of water is added for dissolving), adding 0.2ml of a derivatization reagent 2, 4-dinitrochlorobenzene solution (100 mg to 20ml of 2, 4-dinitrochlorobenzene is taken, acetonitrile is added for diluting to a scale, shaking up is carried out), sealing and capping are carried out, treatment is carried out according to the derivatization conditions listed in the table 2, cooling is carried out, the sample is washed by the solvent for multiple times and is completely transferred into a 10ml measuring bottle, and the sample is obtained after the solvent is diluted to the scale.
Table 2 cyclopropylamine derivatization conditions durability test results
The results show that: levatinib mesylate was derivatized under the varying conditions listed in the Table, with essentially no effect on the assay results, indicating that the derivatization conditions were robust.
Example 5:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding 1ml glacial acetic acid, mixing, adjusting pH to 5.0) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Placing a test sample and a reference substance in a proper amount into a 20ml headspace bottle, adding 2ml of a solvent, adding 0.5ml of a sodium carbonate aqueous solution (taking 2.5g of sodium carbonate, adding 25ml of water for dissolving) and 0.2ml of a derivatization reagent 2, 4-dinitrochlorobenzene solution (taking 100mg of 2, 4-dinitrochlorobenzene to a 20ml measuring bottle, adding acetonitrile for diluting to scales, shaking uniformly), sealing, capping, carrying out water bath at 75 ℃ for 5 hours as derivatization conditions, cooling, washing with the solvent for multiple times, completely transferring into a 10ml measuring bottle, and diluting the solvent to scales to obtain a test sample solution with the concentration of 1.0mg/ml (mevinotinib mesylate); control solution with concentration of 7.5. mu.g/ml.
3) Measurement results
The detection result shows that the product after the derivatization of the cyclopropylamine is single, the separation degree from other peaks is good, and the detection result is shown in a figure 3 and a figure 4.
Example 6:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding 1ml glacial acetic acid, mixing, adjusting pH to 5.0) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Placing a test sample and a reference substance in a proper amount into a 20ml headspace bottle, adding 2ml of a solvent, adding 0.5ml of a sodium carbonate aqueous solution (taking 2.5g of sodium carbonate, adding 25ml of water for dissolving) and 0.2ml of a derivatization reagent 2, 4-dinitrochlorobenzene solution (taking 100mg of 2, 4-dinitrochlorobenzene to a 20ml measuring bottle, adding acetonitrile for diluting to scales, shaking uniformly), sealing, capping, carrying out water bath at 75 ℃ for 5 hours as derivatization conditions, cooling, washing with the solvent for multiple times, completely transferring into a 10ml measuring bottle, and diluting the solvent to scales to obtain a test sample solution with the concentration of 1.0mg/ml (mevinotinib mesylate); control solution with concentration of 7.5. mu.g/ml.
3) Measurement results
The detection result shows that the product after derivatization of the cyclopropylamine is single, and the separation degree from other peaks is good.
Example 7:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding 1ml glacial acetic acid, mixing, adjusting pH to 5.0) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Placing a test sample and a reference substance in a proper amount into a 20ml headspace bottle, adding 2ml of a solvent, adding 0.5ml of a sodium carbonate aqueous solution (taking 2.5g of sodium carbonate, adding 25ml of water for dissolving) and 0.2ml of a derivatization reagent 2, 4-dinitrochlorobenzene solution (taking 100mg of 2, 4-dinitrochlorobenzene to a 20ml measuring bottle, adding acetonitrile for diluting to scales, shaking uniformly), sealing, capping, carrying out water bath at 75 ℃ for 5 hours as derivatization conditions, cooling, washing with the solvent for multiple times, completely transferring into a 10ml measuring bottle, and diluting the solvent to scales to obtain a test sample solution with the concentration of 1.0mg/ml (mevinotinib mesylate); control solution with concentration of 7.5. mu.g/ml.
3) Measurement results
The detection result shows that the product after derivatization of the cyclopropylamine is single, and the separation degree from other peaks is good.
TABLE 3 test results of examples 5 to 7
Example 8:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding glacial acetic acid to adjust pH to 4.0) -acetonitrile-methanol (3:2:1)
Solvent: acetonitrile-water (60:30)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Placing a test sample and a reference substance in a proper amount into a 20ml headspace bottle, adding 2ml of a solvent, adding 0.5ml of a sodium carbonate aqueous solution (taking 2.4g of sodium carbonate, adding 20ml of water for dissolving) and 0.2ml of a derivatization reagent 2, 4-dinitrochlorobenzene solution (taking 100mg of 2, 4-dinitrochlorobenzene to a 20ml measuring bottle, adding acetonitrile for diluting to scales, shaking uniformly), sealing, capping, carrying out water bath at 75 ℃ for 5 hours as derivatization conditions, cooling, washing with the solvent for multiple times, completely transferring into a 10ml measuring bottle, and diluting the solvent to scales to obtain a test sample solution with the concentration of 1.0mg/ml (mevinotinib mesylate); control solution with concentration of 7.5. mu.g/ml.
3) Measurement results
The test results show that the product after derivatization of the cyclopropylamine is single, and the test results are similar to those shown in FIG. 4 in example 5.
Example 9:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding glacial acetic acid 1 to adjust pH to 6.0) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (60:10)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Placing a test sample and a reference substance in a proper amount into a 20ml headspace bottle, adding 2ml of a solvent, adding 0.5ml of a sodium carbonate aqueous solution (taking 2.5g of sodium carbonate, adding 25ml of water for dissolving) and 0.2ml of a derivatization reagent 2, 4-dinitrochlorobenzene solution (taking 100mg of 2, 4-dinitrochlorobenzene to a 20ml measuring bottle, adding acetonitrile for diluting to scales, shaking uniformly), sealing, capping, carrying out water bath at 75 ℃ for 5 hours as derivatization conditions, cooling, washing with the solvent for multiple times, completely transferring into a 10ml measuring bottle, and diluting the solvent to scales to obtain a test sample solution with the concentration of 1.0mg/ml (mevinotinib mesylate); control solution with concentration of 7.5. mu.g/ml.
3) Measurement results
The test results show that the product after derivatization of the cyclopropylamine is single, does not interfere with detection, meets the standard with other peak separation degrees, and is similar to the results shown in fig. 4 in example 5.
Example 10:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.05mol/L ammonium acetate solution (taking 3.85g ammonium acetate, adding 1000ml water to dissolve, adding 1ml glacial acetic acid, mixing, adjusting pH to 5.0) -acetonitrile-methanol (8:1:1)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Placing a test sample and a reference substance in a proper amount into a 20ml headspace bottle, adding 2ml of a solvent, adding 0.5ml of a sodium carbonate aqueous solution (taking 1.6g of sodium carbonate, adding 20ml of water for dissolving) and 0.2ml of a derivatization reagent 2, 4-dinitrochlorobenzene solution (taking 100mg of 2, 4-dinitrochlorobenzene to a 20ml measuring bottle, adding acetonitrile for diluting to scales, shaking uniformly), sealing, capping, carrying out water bath at 75 ℃ for 5 hours as a derivatization condition, cooling, washing with the solvent for multiple times, completely transferring into a 10ml measuring bottle, and diluting the solvent to scales to obtain a test sample solution with the concentration of 1.0mg/ml (mevinotinib mesylate); control solution with concentration of 7.5. mu.g/ml.
3) Measurement results
The detection result shows that the product after the derivatization of the cyclopropylamine is single, the separation degree of the product meets the standard with other peaks, and the test result is similar to the result shown in the figure 4 in the example 5.
Example 11:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.01mol/L phosphate buffer solution (1.36 g potassium dihydrogen phosphate, 1000ml water for dissolution, pH 3.0 adjusted by phosphoric acid) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Precisely weighing an impurity SCR-4338, an impurity SCR-4452, an impurity SCR-4453, an impurity SCR-4342 and a proper amount of the impurities to prepare a solution containing about 7.5mg of each impurity per 1ml, precisely weighing 2ml to a 20ml headspace bottle, adding 10mg of mevalontinib mesylate, precisely weighing 0.5ml of a sodium carbonate aqueous solution (obtained by taking 2.5g of sodium carbonate and adding 25ml of water to dissolve), adding a derivatization reagent 2, 4-dinitrochlorobenzene solution (obtained by taking 100mg to 20ml of 2, 4-dinitrochlorobenzene, adding acetonitrile to dilute to scale and shaking uniformly) 0.2ml, sealing, capping, carrying out water bath at 75 ℃ for 5 hours, cooling, washing with a solvent for multiple times, completely transferring to the 10ml volumetric bottle, and diluting the solvent to scale to prepare an amino-containing impurity reference solution.
3) Measurement results
The detection result shows that the separation degree of the known impurities and the products after the derivatization of the cyclopropylamine under the chromatographic conditions is poor. See fig. 5.
Example 12:
1) chromatographic conditions
The instrument comprises the following steps: high performance liquid chromatograph
A chromatographic column: agilent Zorbax-bones-RP-C18 (150 mm. times.4.6 mm,3.5 μm)
Mobile phase: 0.01mol/L phosphate buffer solution (taking 1.36g potassium dihydrogen phosphate, adding 1000ml water to dissolve, adjusting pH to 6.2 with phosphoric acid) -acetonitrile-methanol (18:11:11)
Solvent: acetonitrile-water (80:20)
Sample introduction amount: 10 μ l
Detection wavelength: 356nm
2) Sample preparation
Precisely weighing an impurity SCR-4338, an impurity SCR-4452, an impurity SCR-4453, an impurity SCR-4342 and a proper amount of the impurities to prepare a solution containing about 7.5mg of each impurity per 1ml, precisely weighing 2ml to a 20ml headspace bottle, adding 10mg of mevalontinib mesylate, precisely weighing 0.5ml of a sodium carbonate aqueous solution (obtained by taking 2.5g of sodium carbonate and adding 25ml of water to dissolve), adding a derivatization reagent 2, 4-dinitrochlorobenzene solution (obtained by taking 100mg to 20ml of 2, 4-dinitrochlorobenzene, adding acetonitrile to dilute to scale and shaking uniformly) 0.2ml, sealing, capping, carrying out water bath at 75 ℃ for 5 hours, cooling, washing with a solvent for multiple times, completely transferring to the 10ml volumetric bottle, and diluting the solvent to scale to prepare an amino-containing impurity reference solution.
3) Measurement results
The detection result shows that the peak type of the product of the known impurity and the derivatization of the cyclopropylamine under the chromatographic condition is poor, and other impurities are suspected to fall on the position of the peak of the cyclopropylamine. See fig. 6.
Claims (12)
1. An HPLC method for detecting cyclopropylamine in lenvatinib mesylate is characterized in that: adopting a reversed phase chromatographic column; performing derivatization treatment on a sample to be detected by using a derivatization reagent; the mobile phase is selected from salt buffer solution containing amine substances and organic solvent.
2. The process of claim 1, wherein the derivatizing agent is selected from the group consisting of 2, 4-dinitrochlorobenzene, 2, 4-dinitrofluorobenzene, benzoyl chloride, and p-nitrophenyl N-succinimidyl acetate.
3. The method of claim 1, wherein the derivatizing step comprises reacting cyclopropylamine with a derivatizing agent to form a cyclopropylamine derivative having ultraviolet absorption.
4. The method according to claim 3, wherein sodium carbonate is added during the derivatization treatment, and the mass ratio of the sodium carbonate to the test sample is 40: 1-60: 1, preferably 50: 1.
5. The method of claim 1, wherein the derivatization time is 1.5 to 12 hours.
6. An HPLC method for detecting cyclopropylamine in lenvatinib mesylate according to claim 1, wherein the temperature of derivatization is 60-80 ℃, preferably 75 ℃.
7. The method according to claim 1, wherein the salt buffer solution is acetate buffer solution or formate buffer solution.
8. The process according to claim 1, wherein the pH of the amine-containing salt buffer solution is 4.0 to 6.0, preferably 5.0.
9. The method according to claim 1, wherein the volume ratio of the amine-containing salt buffer solution to the organic solvent is 1:1 to 1:6, preferably 1:1.2 to 1:4, and more preferably 9: 11.
10. The method according to claim 1, wherein the organic solvent is selected from one or more of acetonitrile, methanol, and ethanol.
11. The method according to claim 1 or claim 4, wherein the organic solvent is preferably acetonitrile and methanol at a volume ratio of 2:1 to 1:3, preferably 1:1, preferably, the detection wavelength is 215-356nm, preferably 356 nm.
12. The method according to claim 1, further comprising dissolving the sample to be tested with a solvent, wherein the solvent comprises acetonitrile and water, and the volume ratio of acetonitrile to water is 2: 1-6: 1, preferably 4: 1.
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| CN105424822A (en) * | 2015-10-29 | 2016-03-23 | 成都百裕科技制药有限公司 | Method for detecting (1R,2S)-2-(3,4-diflurophenyl) cyclopropylamine in ticagrelor |
| CN107305202A (en) * | 2016-04-22 | 2017-10-31 | 北京睿创康泰医药研究院有限公司 | The HPLC methods and impurity that analysis methanesulfonic acid pleasure is cut down for Buddhist nun and its preparation impurity make the purposes of reference standard |
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| CN105424822A (en) * | 2015-10-29 | 2016-03-23 | 成都百裕科技制药有限公司 | Method for detecting (1R,2S)-2-(3,4-diflurophenyl) cyclopropylamine in ticagrelor |
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