CN118050450A - Analysis method of related substances of 2-acylaminothiazole structure - Google Patents
Analysis method of related substances of 2-acylaminothiazole structure Download PDFInfo
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- CN118050450A CN118050450A CN202410384054.6A CN202410384054A CN118050450A CN 118050450 A CN118050450 A CN 118050450A CN 202410384054 A CN202410384054 A CN 202410384054A CN 118050450 A CN118050450 A CN 118050450A
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- 239000000126 substance Substances 0.000 title claims abstract description 15
- 238000004458 analytical method Methods 0.000 title claims abstract description 12
- 239000012535 impurity Substances 0.000 claims abstract description 66
- 238000000034 method Methods 0.000 claims abstract description 40
- FQQOPZRSAIEOGH-UHFFFAOYSA-N 4-(4-chlorothiophen-2-yl)-5-(4-cyclohexylpiperazin-1-yl)-1,3-thiazol-2-amine Chemical compound C1CN(C2CCCCC2)CCN1C=1SC(N)=NC=1C1=CC(Cl)=CS1 FQQOPZRSAIEOGH-UHFFFAOYSA-N 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 102
- 239000000243 solution Substances 0.000 claims description 79
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 53
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 23
- 239000007864 aqueous solution Substances 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 18
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- 238000004587 chromatography analysis Methods 0.000 claims description 9
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- 238000004366 reverse phase liquid chromatography Methods 0.000 abstract 1
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- 238000012360 testing method Methods 0.000 description 14
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
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- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 description 6
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
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- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 3
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- CWXPZXBSDSIRCS-UHFFFAOYSA-N tert-butyl piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCNCC1 CWXPZXBSDSIRCS-UHFFFAOYSA-N 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 2
- -1 4- (4-chlorothiophene-2-yl) -5- (piperazin-1-yl) thiazol-2-amine Chemical compound 0.000 description 2
- NDDIDWDEBBDFIE-UHFFFAOYSA-N 5-bromo-4-(4-chlorothiophen-2-yl)-1,3-thiazol-2-amine Chemical compound ClC=1C=C(SC=1)C=1N=C(SC=1Br)N NDDIDWDEBBDFIE-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- CBVRNADTHTYIAW-UHFFFAOYSA-N tert-butyl 4-[2-amino-4-(4-chlorothiophen-2-yl)-1,3-thiazol-5-yl]piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCN(CC1)c1sc(N)nc1-c1cc(Cl)cs1 CBVRNADTHTYIAW-UHFFFAOYSA-N 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- VSTXCZGEEVFJES-UHFFFAOYSA-N 1-cycloundecyl-1,5-diazacycloundec-5-ene Chemical compound C1CCCCCC(CCCC1)N1CCCCCC=NCCC1 VSTXCZGEEVFJES-UHFFFAOYSA-N 0.000 description 1
- AULWXGLYZIDHCH-UHFFFAOYSA-N 5-bromo-4-(5-bromo-4-chlorothiophen-2-yl)-1,3-thiazol-2-amine Chemical compound BrC1=C(N=C(S1)N)C=1SC(=C(C=1)Cl)Br AULWXGLYZIDHCH-UHFFFAOYSA-N 0.000 description 1
- XUKUURHRXDUEBC-KAYWLYCHSA-N Atorvastatin Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-KAYWLYCHSA-N 0.000 description 1
- XUKUURHRXDUEBC-UHFFFAOYSA-N Atorvastatin Natural products C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CCC(O)CC(O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-UHFFFAOYSA-N 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229960005370 atorvastatin Drugs 0.000 description 1
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- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
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- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to the field of medicine analysis, and provides an analysis method of 2-acylaminothiazole structure related substances, which adopts a high performance liquid phase reversed phase chromatography method, can effectively detect an intermediate 4- (4-chlorothiophene-2-yl) -5- (4-cyclohexylpiperazine-1-yl) thiazole-2-amine and related impurities IM2-B and/or impurities IM2-C therein, and has high separation degree and good durability.
Description
Technical Field
The invention belongs to the field of medicine analysis, and in particular relates to an analysis method of related substances in 4- (4-chlorothienyl-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazole-2-amine.
Background
4- (4-Chlorothiophene-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-amine (hereinafter referred to as IM 2) is an important intermediate in the synthesis of 2-acylaminothiazole compounds, and corresponding reports are made in the compound patent CN 1639157A. Impurities in the product can be introduced into the medicine in a mode of prototype or conductive impurities in a subsequent synthesis process, so that the quality of the medicine is finally influenced, and the medication safety is further influenced, and therefore, related substances in the product are required to be fully researched to ensure the quality controllability. The structural formula of IM2 is shown below:
CN115047106a discloses a method of analyzing IM2 using a high performance liquid chromatograph, but no further measurement study was performed on IM2 related impurities. At present, an analysis method of IM2 and related substances with strong specificity, good precision, high accuracy, excellent separation effect and good durability is urgently needed, and effective support is provided for the production process and quality control.
Disclosure of Invention
The invention aims to provide an analysis method for determining related substances in 4- (4-chlorothiophene-2-yl) -5- (4-cyclohexylpiperazine-1-yl) thiazole-2-amine (IM 2).
The analysis method adopts high performance liquid reverse chromatography, wherein the mobile phase A is 0.0025-0.0075 mol/L ammonium formate aqueous solution, and the mobile phase B is a mixed solution of methanol and acetonitrile.
In some embodiments, the concentration of the aqueous ammonium formate solution in mobile phase A is 0.005mol/L.
In some embodiments, an acid is added to mobile phase a to adjust the pH; preferably, the acid is formic acid or trifluoroacetic acid; preferably, the acid is trifluoroacetic acid; preferably, the volume percentage of the added trifluoroacetic acid is 0.15-0.25%; preferably, the volume percent of trifluoroacetic acid added is 0.2%.
In some embodiments, the volume ratio of methanol to acetonitrile in mobile phase B is 45 to 55; 55-45; preferably, the volume ratio of methanol to acetonitrile is 50:50.
In some embodiments, gradient elution is employed; in some typical embodiments, the gradient elution conditions are:
In some typical embodiments, the gradient elution conditions are:
In some embodiments, the column temperature is 30 to 40 ℃; in some typical embodiments, the column temperature is 35 ℃.
In some embodiments, the flow rate is from 0.7 to 0.9mL/min; in some typical embodiments, the flow rate is 0.8mL/min.
In some embodiments, the chromatographic column uses octadecylsilane chemically bonded silica as a packing; in some typical embodiments, the chromatographic column is Agilent ZORBAX Eclipse Plus, 100mm by 4.6mm,3.5 μm in size.
In some embodiments, the assay is performed on a high performance liquid chromatograph, employing an ultraviolet detector; in some typical embodiments, the assay is performed on a high performance liquid chromatograph using a variable wavelength detector with a detection wavelength of 245-255 nm, preferably 250nm.
In a specific embodiment, a high performance liquid reverse chromatography method is used, a chromatographic column: agilent ZORBAX Eclipse Plus, specification 100mm×4.6mm,3.5 μm;
column temperature: 35 ℃;
Flow rate: 0.8mL/min;
Sample injection amount: 10. Mu.L;
A detector: a variable wavelength detector;
Detection wavelength: 250nm;
Mobile phase a:0.0025 to 0.0075mol/L ammonium formate aqueous solution, adding 0.15 to 0.25 volume percent of trifluoroacetic acid, wherein the volume percent of the trifluoroacetic acid is the volume ratio of the trifluoroacetic acid to the ammonium formate aqueous solution;
Mobile phase B: methanol: acetonitrile=50: 50
The elution gradient conditions were:
In a specific embodiment, a high performance liquid reverse chromatography method is used, a chromatographic column: chromatographic column: agilent ZORBAX Eclipse Plus, specification 100mm×4.6mm,3.5 μm;
column temperature: 35 ℃;
Flow rate: 0.8mL/min;
Sample injection amount: 10. Mu.L;
A detector: a variable wavelength detector;
Detection wavelength: 250nm;
mobile phase a:0.005mol/L ammonium formate aqueous solution, adding 0.2 percent by volume of trifluoroacetic acid, wherein the volume percent of the trifluoroacetic acid is the volume ratio of the volume of the trifluoroacetic acid to the volume of the ammonium formate aqueous solution;
Mobile phase B: methanol: acetonitrile=50: 50
The elution gradient conditions were:
In a specific embodiment, a high performance liquid reverse chromatography method is used, a chromatographic column: chromatographic column: agilent ZORBAX Eclipse Plus, specification 100mm×4.6mm,3.5 μm;
column temperature: 35 ℃;
Flow rate: 0.8mL/min;
Sample injection amount: 10. Mu.L;
A detector: a variable wavelength detector;
Detection wavelength: 250nm;
mobile phase a:0.005mol/L ammonium formate aqueous solution, adding 0.2 percent by volume of trifluoroacetic acid, wherein the volume percent of the trifluoroacetic acid is the volume ratio of the volume of the trifluoroacetic acid to the volume of the ammonium formate aqueous solution;
mobile phase B: methanol: acetonitrile=50: 50;
The elution gradient conditions were:
in some embodiments, the relevant species in IM2 include impurity IM2-B, which has the formula:
in some embodiments, the relevant species in IM2 include impurity IM2-C, which has the formula:
In some embodiments, the relevant species in IM2 include impurities IM2-B or IM2-C, each having the following structural formula:
in some embodiments, the relevant materials in IM2 further include one or more of impurities SM1, IM2-A, having the structure shown below:
on the other hand, the invention also provides the application of the standard reference substance in the method for detecting the impurities of the IM2 in the compound of the formula IM2-B, wherein the structural formula of the compound of the IM2-B is shown as follows:
in yet another aspect, the present invention provides the use of a standard control in a method for detecting impurities in IM2 of a compound of formula IM2-C, the compound of formula IM2-C having the formula:
In yet another aspect, the present invention provides the use of a standard control for IM2-B and/or IM2-C compounds in a method for detecting impurities in IM2, having the following structural formula:
Specifically, all of the 5 detected impurities are process impurities. Wherein SM1 is a residual starting material in the preparation process of IM 2; IM1 is the residue of an intermediate in the preparation process; IM2-A is the reaction product of IM1-A (IM 1-A is dibromo impurity obtained by the reaction of SM1 and NBS, chemical name: 5-bromo-4- (5-bromo-4-chlorothiophene-2-yl) 2-thiazolamine) and SM2, and IM2-B is the product obtained by the reaction of SM2-E and one time of the amount of IM1 impurity. IM2-C is the product of the reaction of SM2-E with IM 1. The preparation route adopted for IM2 is shown below,
The preparation route of SM2 used in the IM2 route preparation diagram is shown below, SM2-E (chemical name: anhydrous piperazine) is SM2-A stripped of BOC impurities,
The invention has the beneficial effects that: the method can effectively detect related substances SM1, IM1 and IM2-A, IM2-B, IM-C in IM2, and the separation degree between the chromatographic peaks and adjacent peaks of the compound is more than 1.5. In particular, the invention discovers the IM2-B and IM2-C compounds, which are all reported for the first time and are brand new compounds. The invention also provides a preparation method of the compound. The analysis method provided by the invention has the advantages of strong specificity, good precision, high accuracy, excellent separation effect and good durability, and can be used for rapidly and accurately carrying out quantitative analysis on related substances in IM2, so that the quality controllability of the atorvastatin maleate and the preparation thereof is ensured, effective support is provided for the production process and quality control thereof, and important reference is provided for the quality control of other medical intermediates or medicines.
Drawings
FIG. 1 is a chromatogram of a system applicability solution of example 3.
FIG. 2 is a chromatogram of the blank solvent of example 3.
FIG. 3 is a chromatogram of the sample solution of example 3.
FIG. 4 is a system applicability solution chromatogram of example 10.
FIG. 5 is a system applicability solution chromatogram of example 11.
FIG. 6 is a partial magnified view of the system applicability solution chromatogram of example 11.
FIG. 7 is a system applicability solution chromatogram for mobile phase A of example 12 as a 0.01mol/L aqueous ammonium formate solution (pH adjusted to 3.8 with formic acid).
FIG. 8 is a system applicability solution chromatogram for mobile phase A of example 12 as a 0.01mol/L aqueous ammonium formate solution (pH adjusted to 3.0 with formic acid).
Detailed Description
The present invention is described more specifically below, but the following examples are not to be construed as limiting the scope of the invention in any way. Acetonitrile (company TEDIA, usa, chromatographic purity); ammonium formate (ROE Co., USA, chromatographic purity); the experimental water is made of ultra-pure water by a merck Millipore ultra-pure water instrument.
The method of the present invention is not limited to the above 5 impurities, any combination of the separation of the above-mentioned impurities and 1 to 5 impurities selected from the above-mentioned impurities by the method of the present invention falls within the scope of the present invention, and any combination of the separation of the above-mentioned impurities and 1 to 5 impurities contained in the above-mentioned impurities by the method of the present invention falls within the scope of the present invention.
The relevant abbreviations correspond to the full names as follows:
1-Boc-piperazine: 1-Boc piperazine
DMF: n, N-dimethylformamide
DBU:1, 8-diazabicyclo undec-7-ene
DCM: dichloromethane (dichloromethane)
NBS: n-bromosuccinimide
Example 1 preparation of impurity IM2-B
Sequentially adding 14.80g of 5-bromo-4- (4-chlorothiophene-2-yl) thiazole-2-amine, 14.00g of 1-Boc-piperazine and 150mL of DMF into a 250mL three-necked flask, dropwise adding 11.40g of DBU, and placing in a water bath at 20-30 ℃ for stirring reaction for 1h; then pouring the reaction solution into a DCM/water system (200 mL of DCM and 100mL of water), stirring and standing, taking an organic layer, washing the organic layer with water twice, and evaporating the organic layer to dryness; the obtained solid was dissolved in 20mL of ethanol, 200mL of n-heptane was added dropwise with stirring to precipitate a solid, which was suction-filtered, and the cake was dried to obtain 18.25g of pale brown solid, which was 4- (2-amino-4- (4-chlorothiophene-2-yl) thiazol-5-yl) piperazine-1-carboxylic acid tert-butyl ester.
2.00G of 4- (2-amino-4- (4-chlorothiophene-2-yl) thiazole-5-yl) piperazine-1-carboxylic acid tert-butyl ester is weighed and added into a 100mL single-mouth bottle, 20mL of DCM and 4mL of TFA are added, stirring is carried out for 1h at 20-30 ℃, then saturated sodium bicarbonate aqueous solution is added dropwise to adjust the pH value to be more than 7, and an organic layer is taken; the aqueous layer was extracted twice with 40ml x 2dcm, the organic layers were combined, purified by column separation after rotary evaporation to dryness, and the eluates containing the desired product were combined and rotary evaporated to dryness to give 1.31g of 4- (4-chlorothiophene-2-yl) -5- (piperazin-1-yl) thiazol-2-amine (as IM 2-B) as a pale brown solid.
MS(ESI)[M+H]+＝301.1;1H NMR(DMSO-d6)：δ7.435-7.432(d,1H),7.363-7.360(d,1H),
7.104(s,2H),3.302-3.283(t,4H),2.968-2.949(t,4H)。
Example 2 preparation of impurity IM2-C
5-Bromo-4- (4-chlorothiophene-2-yl) thiazol-2-amine, piperazine and DMF are added into a 500mL reaction bottle, nitrogen is substituted after stirring, DBU is added, stirring reaction is carried out at room temperature, cooling is carried out to 0-5 ℃ after the reaction is monitored by thin layer chromatography, water/ethanol solution (160 mL/160 mL) is dropwise added under stirring condition, stirring is carried out for 1h under heat preservation, suction filtration is carried out, filter cakes are washed by ethanol, and the filter cakes are dried to obtain 7.70g brown powdery solid, namely impurity 5,5' - (piperazine-1, 4-diyl) bis (4- (4-chlorothiophene-2-yl) thiazol-2-amine) (which is IM 2-C).
The M/z of [ M+H-C 7H5ClN2S2]+ is 301.1, the M/z of [ M-H ] - is 513.0, and the M/z of [ M-H-C 11H13ClN4S2]- is 215.0; 1H NMR(DMSO-d6): delta 7.376-7.374 (d, 4H), 6.879 (s, 4H), 2.988 (s, 8H).
Example 3
Instrument: agilent high performance liquid chromatograph (Agilent, usa);
Chromatographic column: agilent ZORBAX Eclipse Plus (100 mm. Times.4.6 mm,3.5 μm) or equivalent performance chromatographic columns
Column temperature: 35 DEG C
Flow rate: 0.8mL/min
Sample injection amount: 10 mu L
A detector: variable wavelength detector
Detection wavelength: 250nm
Mobile phase a:5mmol/L ammonium formate aqueous solution (ammonium formate 0.31g, water 1000mL was added to dissolve, trifluoroacetic acid 2mL was added)
Mobile phase B: methanol: acetonitrile=50: 50
Elution gradient:
The solution was prepared as follows:
Blank solvent: methanol
Stock solutions of various impurity reference substances: and respectively taking a proper amount of SM1 reference substance, a proper amount of IM2-A reference substance and a proper amount of IM2-B reference substance, precisely weighing, placing into different measuring flasks, adding methanol for dissolving and diluting to prepare a solution containing about 100 mug of each impurity in 1mL, and taking the solution as a stock solution of each impurity reference substance.
IM1 control stock solution: taking a proper amount of the IM1 reference substance, adding methanol for dissolving and diluting to prepare a solution containing about 150 mug of each 1mL of the solution, and taking the solution as an IM1 reference substance stock solution.
IM2-C control stock solution: taking a proper amount of the IM2-C reference substance, adding methanol to dilute the mixture to prepare a solution containing about 50 mug of each 1mL of the solution, and taking the solution as a stock solution of the IM2-C reference substance.
Each impurity localization solution: 1mL of each of SM1, IM2-A, IM2-B, IM-C control stock solutions was taken and placed in 100mL measuring flasks, respectively, diluted to the scale with methanol, and shaken well to obtain each impurity localization solution (SM 1, IM2-A, IM-B concentrations were 1.0. Mu.g/mL, IM1 concentration was 1.5. Mu.g/mL, and IM2-C concentration was 0.5. Mu.g/mL).
Mixing an impurity reference stock solution: 5mL of each of SM1, IM2-A, IM2-B, IM-C control stock solutions are taken and placed in a same 50mL measuring flask, diluted to scale with methanol, and shaken well (the concentration of SM1, IM2-A, IM-B is 10.0 mug/mL, the concentration of IM1 is 15.0 mug/mL, and the concentration of IM2-C is 5.0 mug/mL).
IM2 control stock solution: taking 5mg of IM2 reference substance, precisely weighing, placing into a 50mL measuring flask, adding methanol for dissolving and diluting to prepare a solution containing about 100 mug of each 1mL of the solution, and taking the solution as an IM2 reference substance stock solution.
Standard stock solution: 5mL of each of SM1, IM2-A, IM2-B, IM2-C, IM2 control stock solutions are respectively taken and placed in a same 50mL measuring flask, diluted to scale with methanol, and shaken well (the concentration of each of IM2, SM1 and IM2-A, IM-B is 10.0 mug/mL, the concentration of IM1 is 15.0 mug/mL, and the concentration of IM2-C is 5.0 mug/mL).
System applicability solution: taking about 5mg of IM2 reference substance, placing into a 10mL measuring flask, adding methanol for dissolution, then precisely adding 1mL of mixed impurity reference substance stock solution, diluting to scale with methanol, and shaking to obtain the final product.
Test solution: taking a proper amount of IM2 test sample, precisely weighing, adding methanol for dissolving and diluting to prepare a solution containing 0.5mg of IM2 per 1mL, and taking the solution as a test sample solution.
And (3) measuring: taking 10 mu L of each of the blank solvent, the system applicability solution and the sample solution, injecting into a liquid chromatograph, and recording a chromatogram.
If impurity peaks exist in the chromatogram of the sample solution, the IM2-B, IM2-A peak area is not larger than the main peak area (0.2%) of the control solution, the SM1 is not larger than the main peak area (0.2%) of the control solution according to the corrected peak area (multiplied by the correction factor of 0.67), the IM2-C is not larger than the main peak area (0.1%) of the control solution according to the corrected peak area (multiplied by the correction factor of 0.75), the IM1 is not larger than the main peak area (0.3%) of the control solution according to the corrected peak area (multiplied by the correction factor of 0.61), the peak areas of other single impurities are not larger than 2.5 times (0.5%) of the main peak area of the control solution, and the total impurity amount is not more than 2.0%.
Results:
The chromatogram of the system applicability solution is shown in figure 1, and the peak-outlet sequence is IM2-B (retention time 8.674 min), SM1 (retention time 12.636 min), IM2 (retention time 16.347 min), IM2-A (retention time 21.925 min), IM2-C (retention time 24.683 min) and IM1 (retention time 26.071 min), the separation degree between each two adjacent chromatographic peaks is greater than 1.5, and the base line is stable.
The chromatogram of the blank solvent is shown in fig. 2.
The chromatogram of the sample solution is shown in FIG. 3, the peak time of IM2-A is 21.912min, and IM2-B, SM1 and IM2-C, IM1 are not detected.
EXAMPLE 4 specificity
Precisely measuring 10 μl of the blank solvent, the system applicability solution, the impurity positioning solution and the sample solution of example 3, injecting into a liquid chromatograph, and recording the chromatogram. The test results are shown in Table 1.
Table 1 System applicability results summary table
The result shows that the blank solvent does not interfere with the detection of related substances of the product; in the chromatogram of the system using solution, the minimum separation degree between the chromatographic peaks is 6.6 & gt1.5, the verification requirement is met, the purity angle of the main component peak is 0.258 & ltpurity threshold value 0.288, and the peak purity meets the verification requirement, so the method has good specificity.
Example 5 limit of detection and limit of quantification test
Taking appropriate amounts of the impurity reference substances and the IM2 reference substances according to the response values of the impurity reference substance solutions, precisely weighing, placing into a same measuring flask, adding methanol for dissolving and diluting to scale, and shaking uniformly to enable the peak-to-peak height response values of the components to be basically consistent, and taking the mixture as a stock solution. Taking a proper amount of stock solution, gradually diluting, and taking the stock solution as a quantitative limiting solution according to the signal to noise ratio of more than or equal to 10:1; the signal to noise ratio is more than or equal to 3:1, and the signal to noise ratio is used as a detection limit solution. The test results are shown in tables 2 to 3.
Table 2 summary of limit of detection results
Table 3 summary of quantitative limit results
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From the above test results, it can be seen that: the minimum signal-to-noise ratio of the quantitative concentration limit of each component is more than or equal to 10, the maximum retention time RSD is less than 1.0%, the RSD of the peak area is more than or equal to 10.0% and less than or equal to 10.0%, and the verification requirement is met; the minimum value of the signal to noise ratio of the detection limit solution is 3 or more than 3; the maximum value of the quantitative limit concentration is equal to 0.01 percent less than 0.05 percent of the concentration of the sample, and the verification requirement is met, so that the method can meet the requirement of the detection sensitivity of the IM2 related substances.
EXAMPLE 6 linearity and Range
Taking IM2 and each impurity reference substance, and preparing at least 6 parts of solutions with different concentrations within the range of limiting the quantification to 200% of the limiting concentration for research, wherein the specific operation is as follows:
linear stock solution: the standard stock solution was the same as in example 3.
The linear solution formulation is shown in the following table:
Note that: concentration 1: IM2, SM1, IM2-B, IM-A
Concentration 2: IM1
Concentration 3: IM2-C
Taking appropriate amount of each solution, injecting into a liquid chromatograph, and recording a chromatogram. Linear regression was performed by least squares with the concentration (μg/mL) as x-axis and the peak area as y-axis, and the ratio (k) of the y-axis intercept to the 100% limit concentration was calculated.
The results show that the IM2-B has good linear relation within the range of the limit concentration of 20% -200% in the concentration range of 0.19-1.94 mug/mL, the correlation coefficient r is 0.9999>0.990, the response factor RSD is 2.3% <10.0%, and the y-axis intercept deviation is 1.4% <20%, so as to meet the verification requirement.
SM1 has good linear relation in the range of the limit concentration of 20% -200% within the concentration range of 0.20 μg/mL-2.00 μg/mL, the correlation coefficient r is 0.9998>0.990, the response factor RSD is 2.0% <10%, the y-axis intercept deviation is 1.4% <20%, and the verification requirement is met.
IM2 has good linear relation in the range of the limit concentration of 20% -200% within the concentration range of 0.20 μg/mL-1.98 μg/mL, the correlation coefficient r is 0.9999>0.990, the response factor RSD is 2.1% <10%, and the y-axis intercept deviation is 1.6% <20%, thereby meeting the verification requirement.
The IM2-A has good linear relation within the range of the limit concentration of 20% -200% in the concentration of 0.18 mug/mL-1.83 mug/mL, the correlation coefficient r is 0.9998>0.990, the response factor RSD is 3.4% <10%, the y-axis intercept deviation is 3.0% <20%, and the verification requirement is met.
The linear relation of the IM2-C is good within the range of the limit concentration of 20% -200% in the concentration range of 0.10 mug/mL-0.99 mug/mL, the correlation coefficient r is 0.9998>0.990, the response factor RSD is 1.2% <10%, the y-axis intercept deviation is 1.0% <20%, and the verification requirement is met.
IM1 has good linear relation in the range of the limit concentration of 20% -200% in the concentration range of 0.30 μg/mL-3.02 μg/mL, the correlation coefficient r is 0.9999>0.990, the response factor RSD is 1.9% <10%, and the y-axis intercept deviation is 1.6% <20%, thereby meeting the verification requirement.
From the above experimental results, it can be seen that: the correlation coefficient rmin is 0.9998 & gt0.990, the k value max is 3.0% & lt 20.0%, the RSD of the response factor max is 3.4% & lt 10.0%, and the peak area of each component has good linear relation with the corresponding concentration.
Example 7 correction factor
Taking a proper amount of each reference substance, determining linearity and range according to law in different instruments, recording a chromatogram, and dividing the slope of the impurity by the slope of the main component to obtain the correction factor of the impurity. The results are shown in Table 4.
TABLE 4 correction factor determination results
| Name of the name | E3-B-095 | E3-B-087 | Average value of | Extremely poor |
| IM2-B | 1.01 | 1.02 | 1.02 | 0.01 |
| SM1 | 0.68 | 0.66 | 0.67 | 0.01 |
| IM2 | 1.00 | 1.00 | 1.00 | 0.00 |
| IM2-A | 1.10 | 1.12 | 1.11 | 0.02 |
| IM2-C | 0.76 | 0.74 | 0.75 | 0.01 |
| IM1 | 0.61 | 0.61 | 0.61 | 0.00 |
From the above test results, it can be seen that: the maximum value of correction factors between two different types of instruments is 0.02 < 0.2, the correction factor of impurity IM2-B, IM2-A relative to IM2 is in the range of 0.9-1.1, the correction factors of SM1 and IM2-C, IM1 can be inspected by adopting a self-comparison method without adding correction factors, and the correction factors can be inspected by adopting a self-comparison method with adding correction factors in the range of 0.2-5.
Example 8 accuracy test
Solvent: methanol
Mixing an impurity reference stock solution: the impurity-mixed control stock solution was prepared as in example 3.
Control solution: the preparation method was the same as for the control solution in example 3.
System applicability solution: the system applicability solution under example 3 was taken.
The specific preparation method of the recovery rate solution is shown in the following table:
| Horizontal level | Test article (mg) | Mixed impurity control stock solution volume (mL) | Volumetric flask volume (mL) |
| 50％ | 10 | 1 | 20 |
| 100％ | 10 | 2 | 20 |
| 150％ | 10 | 3 | 20 |
10. Mu.L of each solution was taken and injected into a liquid chromatograph, and the recovery rate was calculated by the self-control method, and the results are shown in tables 5 to 9.
TABLE 5IM2-B accuracy measurement results
TABLE 6SM1 accuracy measurement results
TABLE 7IM2-A accuracy measurement results
TABLE 8IM2-C accuracy measurement results
TABLE 9IM1 accuracy measurement results
The results show that: the recovery rate of water with different concentrations is 97.8% -106.0% calculated by adopting a self-comparison method, the verification requirement of 80% -120% is met, and the maximum value of RSD of the recovery rate of 9 parts of sample solutions with 3 different concentration levels of each impurity is less than 10.0%, so that the accuracy of the method is good by adopting the self-comparison method.
Example 9 durability test
To examine the interference capability of the method itself on variable test factors, we examined the durability of the method, and the examined factors mainly include: column temperature, flow rate, initial ratio, trifluoroacetic acid content, buffer salt concentration (i.e. ammonium formate aqueous solution concentration), methanol ratio in mobile phase, different batch chromatographic columns. The investigation index is the interference condition of the blank solvent on the peak of each impurity and the separation condition of each component in the system applicability solution. The durability chromatographic conditions are shown in table 10 below.
Table 10 durability chromatographic conditions
Note that: YFFX-LC087, YFFX-LC1166 are different batches Agilent ZORBAX Eclipse Plus (100 mm. Times.4.6 mm,3.5 μm) of columns.
Under the condition of each durability, respectively taking a blank solvent, a system applicability solution and 10 mu L of each solution, injecting the solution into a liquid chromatograph, recording a chromatogram, examining the interference condition of the blank solvent on each impurity peak, and the system applicability solution, the separation degree between the chromatographic peaks of each impurity, wherein the test result is shown in table 11.
Table 11 summary of durability results-system applicability solution
| Sequence number | Minimum degree of separation between impurities |
| Standard condition 1 | 5.42 |
| Chromatographic condition 2 | 5.92 |
| Chromatographic condition 3 | 4.51 |
| Chromatographic condition 4 | 5.50 |
| Chromatographic condition 5 | 4.59 |
| Chromatographic condition 6 | 4.88 |
| Chromatographic condition 7 | 5.33 |
| Chromatographic condition 8 | 3.25 |
| Chromatographic condition 9 | 5.50 |
| Chromatographic condition 10 | 5.91 |
| Chromatographic condition 11 | 3.39 |
| Chromatographic condition 12 | 6.18 |
| Chromatographic condition 13 | 4.14 |
| Chromatographic conditions 14 | 5.87 |
Parameters in chromatographic conditions are slightly changed, and when the flow rate (+ -0.1 mL/min), the column temperature (+ -5 ℃) initial proportion (+ -5), the trifluoroacetic acid content (+ -0.05%), the buffer salt concentration (+ -2.5 mmol/L) and the methanol proportion (+ -5%) in a mobile phase are different from those of chromatographic columns of different batches, the minimum separation degree between a main peak, adjacent impurities and the impurities in the chromatograms of the system applicability solution is more than 1.5 under different chromatographic conditions, so that the verification requirement is met, namely the method has good durability.
The inventors have made a search for mobile phase conditions, gradient elution conditions, and the like, see examples 10 to 13 below.
Example 10
Chromatographic column: agilent ZORBAX Eclipse Plus C18 (100 mm. Times.4.6 mm,3.5 μm)
Mobile phase A0.01 mol/L Potassium dihydrogen phosphate solution (1.36 g of Potassium dihydrogen phosphate, 1000mL of water were added to dissolve, and phosphoric acid was added until the pH was 3.8)
Mobile phase B methanol
Flow rate 1.0mL/min
The detection wavelength is 250nm
The column temperature is 35 DEG C
Sample injection volume: 10 mu L
Gradient elution procedure:
The sample formulation is as follows:
Blank solvent: methanol
Positioning solution: and respectively taking a proper amount of SM1, IM1 and IM2-A, IM2-B, IM2-C reference substances, precisely weighing, adding DMSO to dissolve, and diluting with methanol to prepare a solution containing about 1mg of each 1mL of the solution as a positioning solution of each impurity.
System applicability solution: and respectively taking proper amounts of the impurity reference substances and the IM2 reference substances, precisely weighing, adding DMSO for dissolving, and diluting with methanol to prepare a solution containing about 21 mg of IM and 1 mug of each impurity in each 1mL, wherein the solution is used as a system applicability solution.
Test solution: proper amount of IM2 is taken, precisely weighed, dissolved in DMSO and diluted with methanol to prepare a solution containing about 1mg of each 1mL of the solution as a test solution.
Taking a blank solvent and a system applicability solution, respectively 10 mu L of each impurity positioning solution, injecting into a liquid chromatograph, and recording a chromatogram. The test results are shown in Table 12 below, and the corresponding system applicability solution chromatograms are shown in FIG. 4.
Table 12 summary of the separation test results
Results: the blank solvent has no interference to the detection of related substances of the product; in the chromatogram of the system applicability solution (see FIG. 4), the separation degree between IM2-C and the unknown impurity peak is 0.79 < 1.5, and the separation degree cannot meet the requirement.
Example 11
Mobile phase B was changed from methanol to methanol-acetonitrile (50:50) on the basis of the chromatographic conditions of example 10, the other conditions being unchanged.
10 Mu L of the system applicability solution is injected into a liquid chromatograph, a chromatogram is recorded, the test result is shown in table 13, and the corresponding chromatogram is shown in fig. 5.
TABLE 13 summary of separation test results
Results: under the chromatographic conditions, the retention time of IM2 is 19.623min, and the separation degree between each known chromatographic peak and the adjacent chromatographic peak is 1.61 & gt 1.5, namely under the chromatographic conditions, each known component peak can be effectively separated from the adjacent chromatographic peak; however, under chromatographic conditions, the peak-to-peak shape of the unknown impurity was poor at 23.18min (see FIG. 6).
Example 12
Under the chromatographic condition of example 11, the potassium dihydrogen phosphate solution is used as a mobile phase, and the proportion of an organic phase (methanol-acetonitrile) can reach 80% in the gradient elution process, so that the mobile phase A is replaced by an ammonium formate aqueous solution from the potassium dihydrogen phosphate solution, and the mobile phase A is further optimized.
The preparation method comprises the steps of taking 0.63g of ammonium formate respectively, adding 1000mL of water to dissolve the ammonium formate, adding formic acid until the pH value reaches 3.8 or 3.0, taking 10 mu L of system applicability solution in a liquid chromatograph under the same chromatographic conditions as in example 11, and recording a chromatogram.
Results:
The chromatogram of mobile phase A with 0.01mol/L ammonium formate aqueous solution (pH adjusted to 3.8 with formic acid) is shown in FIG. 7, and the peak sequences are IM2-B (retention time 9.813 min), IM2 (retention time 17.481 min), SM1 (retention time 19.852 min), IM2-A (retention time 27.071 min), IM2-C (retention time 27.734 min), IM1 (retention time 32.594 min). The separation degree of the IM2 and the unknown impurities (retention time 18.529 min) is less than 1.5, and the separation degree cannot meet the requirement.
The chromatogram of mobile phase A with 0.01mol/L ammonium formate aqueous solution (pH adjusted to 3.0 with formic acid) is shown in FIG. 8, and the peak sequences are IM2-B (retention time 9.602 min), SM1 (retention time 13.486 min), IM2 (retention time 17.445 min), IM2-A (retention time 22.713 min), IM2-C (retention time 27.499 min), IM1 (retention time 32.766 min). The separation degree of unknown impurities (retention time 19.688 min) is poor, and accurate quantification is inconvenient; and the baseline is not stable, and accurate integration of impurities is difficult.
Example 13
Chromatographic column: agilent ZORBAX Eclipse Plus (100 mm. Times.4.6 mm,3.5 μm);
Detection wavelength: 250nm;
column temperature: 35 ℃; injector temperature: 4 ℃;
Flow rate: 1.0mL/min;
Mobile phase a:0.01mol/L ammonium formate aqueous solution (0.63 g of ammonium formate, 1000mL of water are added to dissolve, and formic acid is added until the pH value is 3.0);
mobile phase B: methanol: acetonitrile=50: 50;
Gradient elution was performed as follows:
the solution was prepared and measured in the same manner as in example 10.
Results: the degree of separation between the impurities may be less than 1.5, and thus, the impurities may not be separated effectively.
Claims (10)
1. A method for analyzing related substances in 4- (4-chlorothien-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazole-2-amine is characterized in that: high performance liquid reverse chromatography is adopted, wherein the mobile phase A is 0.0025-0.0075 mol/L ammonium formate aqueous solution, and the mobile phase B is a mixed solution of methanol and acetonitrile.
2. The method according to claim 1, wherein the concentration of the aqueous ammonium formate solution in mobile phase a is 0.005mol/L.
3. The method according to claim 1, wherein an acid is added to the mobile phase a to adjust the pH; preferably, the acid is formic acid or trifluoroacetic acid; preferably, the acid is trifluoroacetic acid; preferably, the volume percentage of the added trifluoroacetic acid is 0.15-0.25%; preferably, the volume percent of trifluoroacetic acid added is 0.2%.
4. The process according to claim 1, wherein the volume ratio of methanol to acetonitrile in mobile phase B is 45-55; 55-45; preferably, the volume ratio of methanol to acetonitrile is 50:50.
5. The method of claim 1, wherein the high performance liquid reverse chromatography employs gradient elution conditions of:
；
Preferably, the gradient elution conditions are:
；
in the elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of mobile phase A refers to the percentage of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent.
6. The method according to claim 1, wherein the column temperature of the high performance liquid reverse chromatography is 30-40 ℃; preferably, the column temperature is 35 ℃; the flow rate of the high performance liquid reverse chromatography is 0.7-0.9 mL/min; preferably, the flow rate is 0.8mL/min; the chromatographic column of the high performance liquid reverse chromatography adopts octadecylsilane chemically bonded silica gel as filler; preferably, the chromatographic column is Agilent ZORBAX Eclipse Plus, specification 100mm x 4.6mm,3.5 μm; the analysis method is carried out on a high performance liquid chromatograph and an ultraviolet detector is adopted; preferably, the analysis method is performed on a high performance liquid chromatograph, a variable wavelength detector is adopted, and the detection wavelength is 245-255 nm; preferably, the detection wavelength is 250nm.
7. The method of claim 1, wherein the column of high performance liquid chromatography: agilent ZORBAX Eclipse Plus, specification 100mm×4.6mm,3.5 μm;
column temperature: 35 ℃;
Flow rate: 0.8mL/min;
Sample injection amount: 10. Mu.L;
A detector: a variable wavelength detector;
Detection wavelength: 250nm;
Mobile phase a:0.0025 to 0.0075mol/L ammonium formate aqueous solution, adding 0.15 to 0.25 volume percent of trifluoroacetic acid, wherein the volume percent of the trifluoroacetic acid is the volume ratio of the trifluoroacetic acid to the ammonium formate aqueous solution;
mobile phase B: methanol: acetonitrile=50: 50;
The elution gradient conditions were:
；
in the elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of mobile phase A refers to the percentage of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent.
8. The method of claim 1, wherein the column of high performance liquid chromatography: agilent ZORBAX Eclipse Plus, specification 100mm×4.6mm,3.5 μm;
column temperature: 35 ℃;
Flow rate: 0.8mL/min;
Sample injection amount: 10. Mu.L;
A detector: a variable wavelength detector;
Detection wavelength: 250nm;
mobile phase a:0.005mol/L ammonium formate aqueous solution, adding 0.2 percent by volume of trifluoroacetic acid, wherein the volume percent of the trifluoroacetic acid is the volume ratio of the volume of the trifluoroacetic acid to the volume of the ammonium formate aqueous solution;
mobile phase B: methanol: acetonitrile=50: 50;
The elution gradient conditions were:
；
in the elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of mobile phase A refers to the percentage of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent.
9. Use of a compound of formula IM2-B in a standard control for a method of detecting 4- (4-chlorothien-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-amine impurities, the compound of formula IM2-B having the formula:
10. Use of a standard control of a compound of formula IM2-C in a method for detecting 4- (4-chlorothien-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-amine impurities, the structural formula of the IM2-C compound being as follows:
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