CN117538450A - Detection method of bazedoxifene acetate intermediate - Google Patents
Detection method of bazedoxifene acetate intermediate Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 49
- OMZAMQFQZMUNTP-UHFFFAOYSA-N acetic acid;1-[[4-[2-(azepan-1-yl)ethoxy]phenyl]methyl]-2-(4-hydroxyphenyl)-3-methylindol-5-ol Chemical compound CC(O)=O.C=1C=C(OCCN2CCCCCC2)C=CC=1CN1C2=CC=C(O)C=C2C(C)=C1C1=CC=C(O)C=C1 OMZAMQFQZMUNTP-UHFFFAOYSA-N 0.000 title claims abstract description 30
- 229960003713 bazedoxifene acetate Drugs 0.000 title claims abstract description 27
- KRIJKJMYOVWRSJ-UHFFFAOYSA-N 3-methyl-5-phenylmethoxy-2-(4-phenylmethoxyphenyl)-1h-indole Chemical compound C1=C2C(C)=C(C=3C=CC(OCC=4C=CC=CC=4)=CC=3)NC2=CC=C1OCC1=CC=CC=C1 KRIJKJMYOVWRSJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 78
- 238000000034 method Methods 0.000 claims description 31
- 238000010828 elution Methods 0.000 claims description 25
- 239000012488 sample solution Substances 0.000 claims description 23
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 11
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 4
- 230000002378 acidificating effect Effects 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 64
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 13
- 238000002360 preparation method Methods 0.000 abstract description 13
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 66
- 239000002904 solvent Substances 0.000 description 17
- 238000005303 weighing Methods 0.000 description 14
- 238000007865 diluting Methods 0.000 description 13
- 239000013558 reference substance Substances 0.000 description 13
- 239000011550 stock solution Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 230000004807 localization Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000012490 blank solution Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 208000033830 Hot Flashes Diseases 0.000 description 1
- 206010060800 Hot flush Diseases 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 229960000817 bazedoxifene Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000009802 hysterectomy Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 208000001685 postmenopausal osteoporosis Diseases 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229910052611 pyroxene Inorganic materials 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001457 vasomotor Effects 0.000 description 1
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
- G01N30/02—Column chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J20/287—Non-polar phases; Reversed phases
-
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
-
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- 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
-
- 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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
Abstract
The invention provides a detection method of bazedoxifene acetate intermediate (3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole), which adopts high performance liquid chromatography to detect and analyze bazedoxifene acetate intermediate and various impurities, can effectively elute, separate and quantitatively detect each component, and has the advantages of good sensitivity, strong specificity and high accuracy, thereby realizing scientific monitoring of the preparation process of bazedoxifene acetate, the quality of bulk drugs and related preparation products.
Description
Technical Field
The invention relates to a detection method of bazedoxifene acetate intermediate, in particular to a detection method of 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole and related substances thereof.
Background
Bazedoxifene acetate is a small molecule drug developed by wheatstone and later purchased by pyroxene. Bazedoxifene acetate was first marketed in italy and spain by approval of the european medical agency (EMEA) at month 4 of 2009 under the trade name Conbriza; in 2010, 7 is marketed in Japan under the trade name Viviant; the 10 th month of 2013 was approved by the united states Food and Drug Administration (FDA) and marketed under the trade name duave in the united states for use in postmenopausal women without hysterectomy, to treat moderate to severe climacteric related vasomotor symptoms (hot flashes) and to prevent postmenopausal osteoporosis.
Bazedoxifene acetate having the chemical name 1- [4- (2-azepan-1-yl-ethoxy-benzyl)]-2- (4-hydroxy-phenyl) -3-methyl-1H-indol-5-ol acetate, english name Bazedoxifene Acetate, CAS number 198481-33-3, molecular formula C 32 H 38 N 2 O 5 The molecular weight is 530.67, and the chemical structure is as follows:
3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole is an important intermediate for preparing bazedoxifene acetate, CAS number is 198479-63-9, molecular formula is C 29 H 25 NO 2 The molecular weight is 419.53, and the chemical structure is as follows:
in the synthesis reaction of bazedoxifene acetate, the impurities in the intermediate are inevitably carried into bazedoxifene acetate, and the quality, safety and effectiveness of bazedoxifene acetate are directly influenced, so that the intermediate is required to be analyzed and detected to control the impurity content, and no effective detection method for related substances of the intermediate exists in the prior art.
Disclosure of Invention
The invention aims to: the invention aims to provide a detection method of a bazedoxifene acetate intermediate 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole related substance.
The technical scheme is as follows: the invention relates to a detection method of bazedoxifene acetate intermediate 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole, which comprises the following steps:
(1) Preparing a sample;
(2) And (3) detection: adopting octadecylsilane chemically bonded silica as a reversed phase chromatographic column of a filler, taking an acidic buffer solution with the volume ratio of 0.05-0.5% as a mobile phase A, and taking an organic solvent as a mobile phase B for gradient elution; wherein the gradient elution procedure is as follows:
a is more than 0 and less than or equal to 5 minutes, B is more than 5 and less than or equal to 20 minutes, C is more than 20 and less than or equal to 26 minutes, D is more than 26 and less than or equal to 30 minutes, E is more than 30 and less than or equal to 35 minutes;
(3) Analysis: and recording a chromatogram, and calculating the content of each detection component.
Wherein, the detection components in the step (3) are as follows:
preferably, the gradient elution procedure described in step (2) is as follows:
further preferably, the gradient elution procedure described in step (2) is as follows:
preferably, mobile phase A in step (2) is 0.1% acidic buffer and mobile phase B is acetonitrile.
It is further preferred that the mobile phase a in step (2) is selected from the group consisting of aqueous trifluoroacetic acid, aqueous formic acid, aqueous phosphoric acid and aqueous perchloric acid, more preferably aqueous phosphoric acid.
Preferably, the reversed phase chromatographic column in the step (2) has a filler particle size of 3.0-5.0 μm, a chromatographic column length of 100-150 mm and a chromatographic column diameter of 2.0-4.6 mm.
Further preferably, the reversed phase chromatographic column in the step (2) has a packing particle size of 3.0 μm, a chromatographic column length of 150mm and a chromatographic column diameter of 4.6mm; more preferably PhenomnexC18 (4.6 mm. Times.150 mm,3.5 μm) or an equivalent thereof.
Preferably, the flow rate of the mobile phase in the step (2) is 0.8-1.2 ml/min, more preferably 1.0ml/min; the column temperature of the chromatographic column is 25-30 ℃, more preferably 25 ℃; the detection wavelength is 210nm to 230nm, more preferably 220nm.
Preferably, the concentration of the bazedoxifene acetate intermediate sample solution in step (1) is 0.1-1.0 mg/ml, more preferably 0.5mg/ml.
Specifically, the sample prepared in the step (1) comprises a test sample solution, a bazedoxifene acetate intermediate positioning solution, various impurity positioning solutions and a system applicability solution.
The preparation method of the bazedoxifene acetate intermediate test sample solution comprises the following steps:
precisely weighing a bazedoxifene acetate intermediate to be tested, dissolving in a solvent, and diluting to a target concentration to obtain a test solution.
The preparation method of the intermediate and the positioning solution of each impurity is as follows:
intermediate positioning solution: weighing 25mg of intermediate reference substance, adding acetonitrile for dissolving and quantitatively diluting to prepare a solution containing about 0.5mg of intermediate reference substance per 1ml, and taking the solution as an intermediate stock solution; then, the solution was quantitatively diluted to about 10. Mu.g per 1ml to prepare a positioning solution.
Impurity 1 localization solution: weighing 15mg of impurity 1 reference substance, dissolving in solvent, and quantitatively diluting to obtain solution containing about 0.3mg of impurity 1 per 1 ml; then, the solution was quantitatively diluted to about 6. Mu.g per 1ml, and used as an impurity 1-localized solution.
Impurity 2 localization solution: weighing 15mg of impurity 2 reference substance, dissolving in solvent, and quantitatively diluting to obtain solution containing about 0.3mg of impurity 2 per 1 ml; then, the solution was quantitatively diluted to about 6. Mu.g per 1ml, and used as an impurity 2-localized solution.
Impurity 3 localization solution: weighing 15mg of impurity 3 reference substance, dissolving in solvent, and quantitatively diluting to obtain solution containing about 0.3mg of impurity 3 per 1 ml; then, the solution was quantitatively diluted to about 6. Mu.g per 1ml, and used as an impurity 3-localized solution.
The preparation method of the system applicability solution comprises the following steps:
1ml each of the intermediate and impurity-locating solution was removed, and diluted with acetonitrile into a 50ml measuring flask as a system-applicable solution.
Acetonitrile used in the above formulation is a diluent and may optionally be replaced with any other suitable solvent.
The content calculation method of each detection component in the step (3) preferably adopts an area normalization method and an external standard method.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
the 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole and impurities thereof can be effectively eluted, separated and quantitatively detected, and the method has the advantages of good sensitivity, strong specificity and high accuracy, and can be used for scientifically monitoring the preparation process of bazedoxifene acetate, the quality of bulk drugs and related preparation products.
Drawings
FIG. 1 is an HPLC detection chromatogram of example 1 of the present invention;
FIG. 2 is a HPLC detection chromatogram of example 2 of the present invention;
FIG. 3 is a HPLC detection chromatogram of example 3 of the present invention;
FIG. 4 is a HPLC detection chromatogram of the system applicability solution in example 4 of the present invention;
FIG. 5 is a HPLC detection chromatogram of the sample solution of example 4;
FIG. 6 is a HPLC detection chromatogram of the detection limit;
FIG. 7 is a quantitative limit HPLC detection chromatogram;
FIG. 8 is a HPLC detection chromatogram for 0 hours of solution stability;
FIG. 9 is a HPLC detection chromatogram for 17 hours of solution stability;
FIG. 10 is an HPLC detection chromatogram with a durability of 0.8ml/min flow rate;
FIG. 11 is a HPLC detection chromatogram for a 30℃column temperature for durability;
fig. 12 is an HPLC detection chromatogram with an initial ratio of durable mobile phase a at 88%.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples.
Example 1
1. Chromatographic conditions
Chromatographic column: phenomenexC18(4.6mm×150mm，3.5μm)；
A detector: a Diode Array Detector (DAD);
detection wavelength: 220nm;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
mobile phase: mobile phase a:0.1% phosphoric acid solution; mobile phase B: acetonitrile;
solvent: acetonitrile;
gradient elution procedure: see table 1;
TABLE 1 gradient elution procedure of example 1
| Elution time (minutes) | Mobile phase A% | Mobile phase B% |
| 0 | 95 | 5 |
| 5 | 30 | 70 |
| 20 | 0 | 100 |
| 26 | 0 | 100 |
| 26.1 | 95 | 5 |
| 35 | 95 | 5 |
Sample injection volume: 5 μl.
2. Preparation of sample solutions
Intermediate positioning solution: precisely weighing 25mg of 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole reference substance, placing into a 50ml measuring flask, adding 5ml of acetonitrile, diluting to a fixed volume to a scale, shaking uniformly, and taking the mixture as a 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole positioning solution.
3. Detection and results
Acetonitrile used in the above formulation is a diluent and may optionally be replaced with any other suitable solvent.
As a result, referring to fig. 1, it can be seen that rt= 12.819min is the peak of bazedoxifene acetate intermediate in the present example, and the main peak is trailing in the detection method.
Example 2
1. Chromatographic conditions
Chromatographic column: phenomenexC18(4.6mm×150mm，3.5μm)；
A detector: a Diode Array Detector (DAD);
detection wavelength: 220nm;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
mobile phase: mobile phase a:0.1% phosphoric acid solution; mobile phase B: acetonitrile;
solvent: acetonitrile;
gradient elution procedure: see table 2;
TABLE 2 gradient elution procedure of example 2
| Elution time (minutes) | Mobile phase A% | Mobile phase B% |
| 0 | 70 | 30 |
| 5 | 30 | 70 |
| 20 | 0 | 100 |
| 26 | 0 | 100 |
| 26.1 | 70 | 30 |
| 35 | 70 | 30 |
Sample injection volume: 5 μl.
2. Preparation of sample solutions
Intermediate positioning solution: precisely weighing 15mg of 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole reference substance, placing into a 50ml measuring flask, adding 5ml of acetonitrile, diluting to constant volume to scale, shaking uniformly, and taking as stock solution; 1.0ml of stock solution is precisely removed, placed in a 50ml measuring flask, diluted to a scale by adding a solvent, and shaken uniformly to obtain a 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole positioning solution.
Impurity 1 localization solution: precisely weighing 15mg of impurity 1 reference substance, placing into a 50ml measuring flask, adding acetonitrile-water (10:90) 5ml, diluting to constant volume to scale, shaking uniformly, and taking as impurity stock solution of impurity 1; and precisely transferring 1.0ml of impurity 1 impurity stock solution, placing into a 50ml measuring flask, adding solvent to dilute to scale, shaking uniformly, and taking as impurity 1 positioning solution.
Impurity 2 localization solution: precisely weighing 15mg of impurity 2 reference substance, placing into a 50ml measuring flask, adding 5ml of acetonitrile, diluting to constant volume to scale, shaking uniformly, and taking as impurity stock solution of impurity 2; and precisely transferring 1.0ml of impurity 2 impurity stock solution, placing into a 50ml measuring flask, adding solvent to dilute to scale, shaking uniformly, and taking the solution as impurity 2 positioning solution.
Impurity 3 localization solution: precisely weighing 15mg of impurity 3 reference substance, placing into a 50ml measuring flask, adding 5ml of acetonitrile, diluting to constant volume to scale, shaking uniformly, and taking as impurity stock solution of impurity 3; and precisely transferring 1.0ml of impurity 3 impurity stock solution, placing into a 50ml measuring flask, adding solvent to dilute to scale, shaking uniformly, and taking the solution as impurity 3 positioning solution.
Blank solution: and (3) a solvent.
System applicability solution: 1ml each of the intermediate and impurity-locating solution was removed, and diluted with acetonitrile into a 50ml measuring flask as a system-applicable solution.
3. Detection and results
And respectively measuring 5 mu l of each of the blank solution, the impurity positioning solution and the system applicability solution, and respectively injecting into a liquid chromatograph.
As a result, referring to fig. 2, it can be seen that in this example, rt= 12.735min is a peak of bazedoxifene acetate intermediate, rt=3.575 min is a peak of impurity 1, rt= 9.280min is a peak of impurity 2, and rt= 9.378min is a peak of impurity 3, and the separation degree of impurity 2 from impurity 3 is 0.78, so that baseline separation cannot be achieved.
Example 3
1. Chromatographic conditions
Chromatographic column: phenomenexC18(4.6mm×150mm，3.5μm)；
A detector: a Diode Array Detector (DAD);
detection wavelength: 220nm;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
mobile phase: mobile phase a:0.1% phosphoric acid solution; mobile phase B: acetonitrile;
solvent: acetonitrile;
gradient elution procedure: see table 3;
TABLE 3 gradient elution procedure of example 3
Sample injection volume: 5 μl.
2. Preparation of sample solutions
Sample solutions were prepared in the same manner as in example 2.
3. Detection and results
And respectively measuring 5 mu l of each of the blank solution, the impurity positioning solution and the system applicability solution, and respectively injecting into a liquid chromatograph.
As a result, referring to fig. 3, it can be seen that in this example, rt= 13.003min is a peak of bazedoxifene acetate intermediate, rt= 4.111min is a peak of impurity 1, rt= 9.381min is a peak of impurity 2, and rt= 9.490min is a peak of impurity 3, and the separation degree of impurity 2 from impurity 3 is 0.91, so that baseline separation cannot be achieved.
Example 4: specificity test
1. Chromatographic conditions
Chromatographic column: phenomenexC18(4.6mm×150mm，3.5μm)；
A detector: a Diode Array Detector (DAD);
detection wavelength: 220nm;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
mobile phase: mobile phase a:0.1% phosphoric acid solution; mobile phase B: acetonitrile;
solvent: acetonitrile;
gradient elution procedure: see table 4;
TABLE 4 gradient elution procedure of example 4
| Elution time (minutes) | Mobile phase A% | Mobile phase B% |
| 0 | 85 | 15 |
| 5 | 30 | 70 |
| 20 | 0 | 100 |
| 26 | 0 | 100 |
| 26.1 | 85 | 15 |
| 35 | 85 | 15 |
Sample injection volume: 5 μl.
2. Preparation of sample solutions
Test solution: precisely weighing 25mg of 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole reference substance, placing into a 50ml measuring flask, adding acetonitrile 5ml, diluting to constant volume to scale, shaking uniformly to obtain 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole sample solution;
other sample solutions were prepared in the same manner as in example 2.
3. Detection and results
And respectively weighing 5 mu l of blank solution, impurity positioning solution, system applicability solution and sample solution, and respectively injecting into a liquid chromatograph.
The test results are shown in figures 4-5 and table 5, and the separation degree between the impurities and the main component is more than or equal to 1.5; the blank solution had no interference at the retention time of the main peak in the test sample solution.
TABLE 5 results of specificity investigation
Example 5: limit of detection and limit of quantification
The system applicability solution prepared in the embodiment 2 is diluted step by step to a proper multiple, and the solution with the signal to noise ratio more than or equal to 10:1 is taken as a quantitative limiting solution; and taking the solution with the signal to noise ratio more than or equal to 3:1 as a detection limit solution.
Respectively measuring 5 μl of each solution precisely, injecting into a liquid chromatograph, detecting and limiting solution sample injection 1 needle, quantitatively limiting solution continuous sample injection 6 needle according to the gradient elution procedure in example 4, wherein the detection results are shown in chromatograms 6-7, and the calculation results are shown in tables 6-7.
TABLE 6 quantitative Limit verification results
| Name of the name | Concentration (μg/ml) | S/N | Sensitivity (%) |
| Impurity 1 | 1.2016 | 43.7 | 0.2 |
| Impurity 2 | 1.2384 | 24.9 | 0.2 |
| Impurity 3 | 1.2280 | 14.0 | 0.2 |
| Intermediate products | 1.2312 | 31.8 | 0.2 |
TABLE 7 detection limit verification results
| Name of the name | Concentration (μg/ml) | S/N | Sensitivity (%) |
| Impurity 1 | 0.12016 | 3.4 | 0.02 |
| Impurity 2 | 0.12384 | 10.8 | 0.02 |
| Impurity 3 | 0.12280 | 7.2 | 0.02 |
| Intermediate products | 0.12312 | 9.9 | 0.02 |
Example 6: solution stability
1. Chromatographic conditions
Chromatographic column: phenomenexC18(4.6mm×150mm，3.5μm)；
A detector: a Diode Array Detector (DAD);
detection wavelength: 220nm;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
mobile phase: mobile phase a:0.1% phosphoric acid solution; mobile phase B: acetonitrile;
solvent: acetonitrile;
gradient elution procedure: see table 8;
TABLE 8 gradient elution procedure of example 6
| Elution time (minutes) | Mobile phase A% | Mobile phase B% |
| 0 | 85 | 15 |
| 5 | 30 | 70 |
| 20 | 0 | 100 |
| 26 | 0 | 100 |
| 26.1 | 85 | 15 |
| 35 | 85 | 15 |
Sample injection volume: 5 μl.
2. Preparation of sample solutions
Test solution: precisely weighing 25mg of 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole reference substance, placing into a 50ml measuring flask, adding acetonitrile 5ml, diluting to constant volume to scale, shaking uniformly to obtain 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole sample solution;
sampling the sample solution according to the gradient elution program at 0h, 3h, 5h, 8h, 14h and 17h respectively, wherein the known impurity content, the other single impurity (maximum) content and the total impurity content RSD in the sample solution are less than or equal to 6.0%, and the number of the impurities is basically unchanged; the retention time RSD% of the sample solution in 17h is 0.04%, and the peak area RSD% is 0.61%, which meets the requirements, and shows that the stability of the solution is good. The detection results are shown in chromatograms 8-9, and the calculation results are shown in Table 9.
TABLE 9 System applicability solution stability investigation results
Example 7: durability of
1. Chromatographic conditions
Chromatographic column: phenomenexC18(4.6mm×150mm，3.5μm)；
A detector: a Diode Array Detector (DAD);
detection wavelength: 220nm;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
mobile phase: mobile phase a:0.1% phosphoric acid solution; mobile phase B: acetonitrile;
solvent: acetonitrile;
gradient elution procedure: see table 10;
TABLE 10 gradient elution procedure of example 7
Sample injection volume: 5 μl.
2. Preparation of sample solutions
Test solution: precisely weighing 25mg of 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole reference substance, placing into a 50ml measuring flask, adding acetonitrile 5ml, diluting to constant volume to scale, shaking uniformly to obtain 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole sample solution;
other sample solutions were prepared in the same manner as in example 2.
Durability test conditions the flow rate of the foregoing conditions was adjusted to 0.8ml/min and the column temperature was adjusted to 30 ℃; the initial proportion of the mobile phase A is adjusted to 88%, sample solutions are respectively fed, chromatographic detection results are shown in the chromatograms 10-12, and it can be seen that the initial proportion of the mobile phase A in the method is 83-88%, the flow rate of the mobile phase is 0.8-1.2 ml/min, the column temperature of the chromatographic column is 25-30 ℃, and the method is applicable and meets the requirement of durability.
From the above experiments, the detection method provided by the embodiment 4 of the invention has the advantages of strong specificity, wide linear range, high sensitivity, high accuracy, good solution stability and durability, and can be used for detecting bazedoxifene acetate intermediate samples and impurities.
Claims (10)
1. The detection method of the bazedoxifene acetate intermediate is 3-methyl-5-benzyloxy-2- (4-benzyloxy phenyl) -1H-indole, and is characterized by comprising the following steps of:
(1) Preparing a sample;
(2) And (3) detection: adopting octadecylsilane chemically bonded silica as a reversed phase chromatographic column of a filler, taking an acidic buffer solution with the volume ratio of 0.05-0.5% as a mobile phase A, and taking an organic solvent as a mobile phase B for gradient elution; wherein the gradient elution procedure is as follows:
a is more than 0 and less than or equal to 5 minutes, B is more than 5 and less than or equal to 20 minutes, C is more than 20 and less than or equal to 26 minutes, D is more than 26 and less than or equal to 30 minutes, E is more than 30 and less than or equal to 35 minutes;
(3) Analysis: and recording a chromatogram, and calculating the content of each detection component.
2. The method according to claim 1, wherein the detection composition in step (3) is as follows:
3. the method according to claim 1 or 2, wherein the gradient elution procedure of step (2) is as follows:
4. a method according to claim 3, wherein the gradient elution procedure of step (2) is as follows:
5. the method according to claim 1 or 2, wherein mobile phase a in step (2) is 0.1% acidic buffer and mobile phase B is acetonitrile.
6. The method according to claim 5, wherein the mobile phase A in the step (2) is selected from the group consisting of aqueous trifluoroacetic acid, aqueous formic acid, aqueous phosphoric acid and aqueous perchloric acid.
7. The method according to claim 1 or 2, wherein the reversed-phase column in step (2) has a packing particle size of 3.0 to 5.0 μm, a column length of 100 to 150mm, and a column diameter of 2.0 to 4.6mm.
8. The method according to claim 8, wherein the reversed-phase column in step (2) has a packing particle size of 3.0 μm, a column length of 150mm, and a column diameter of 4.6mm.
9. The method according to claim 1 or 2, wherein the flow rate of the mobile phase in the step (2) is 0.8-1.2 ml/min, the column temperature of the chromatographic column is 25-30 ℃, and the detection wavelength is 210-230 nm.
10. The method according to claim 1 or 2, wherein the concentration of the bazedoxifene acetate intermediate test sample solution in step (1) is 0.1-1.0 mg/ml.
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