CN117630260A - Method for detecting isomer in aryl propionic acid compound - Google Patents
Method for detecting isomer in aryl propionic acid compound Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 46
- -1 aryl propionic acid compound Chemical class 0.000 title claims abstract description 19
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 title description 24
- 235000019260 propionic acid Nutrition 0.000 title description 12
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 238000000926 separation method Methods 0.000 claims abstract description 26
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 12
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- 150000001875 compounds Chemical class 0.000 claims description 61
- 239000012488 sample solution Substances 0.000 claims description 50
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- 238000002360 preparation method Methods 0.000 claims description 37
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 24
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 20
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 16
- 239000000523 sample Substances 0.000 claims description 16
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- 238000002347 injection Methods 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 12
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- 239000012085 test solution Substances 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims description 2
- SHAHPWSYJFYMRX-GDLCADMTSA-N (2S)-2-(4-{[(1R,2S)-2-hydroxycyclopentyl]methyl}phenyl)propanoic acid Chemical compound C1=CC([C@@H](C(O)=O)C)=CC=C1C[C@@H]1[C@@H](O)CCC1 SHAHPWSYJFYMRX-GDLCADMTSA-N 0.000 abstract description 4
- 150000003839 salts Chemical class 0.000 abstract description 4
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- GOFLNFPIFZZLIP-UHFFFAOYSA-N ethanol;2,2,2-trifluoroacetic acid Chemical compound CCO.OC(=O)C(F)(F)F GOFLNFPIFZZLIP-UHFFFAOYSA-N 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 238000010828 elution Methods 0.000 description 8
- 229960002373 loxoprofen Drugs 0.000 description 8
- BAZQYVYVKYOAGO-UHFFFAOYSA-M loxoprofen sodium hydrate Chemical compound O.O.[Na+].C1=CC(C(C([O-])=O)C)=CC=C1CC1C(=O)CCC1 BAZQYVYVKYOAGO-UHFFFAOYSA-M 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 6
- 239000002207 metabolite Substances 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- IDONOSNYFLNWQN-UHFFFAOYSA-N hexane;methanol;propan-2-ol Chemical compound OC.CC(C)O.CCCCCC IDONOSNYFLNWQN-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- GCFHZZWXZLABBL-UHFFFAOYSA-N ethanol;hexane Chemical compound CCO.CCCCCC GCFHZZWXZLABBL-UHFFFAOYSA-N 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 2
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- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to a method for detecting isomers in aryl propionic acid compounds. The method adopts high performance liquid chromatography detection, can completely separate 8 isomers, and has the separation degree of each chromatographic peak not smaller than 1.5, thereby effectively controlling the quality of the loxoprofen active metabolite and the salt thereof, more effectively realizing quality monitoring on the loxoprofen active metabolite and the salt thereof, and having practical guiding significance for improving the quality of related products.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, relates to a method for detecting isomers in aryl propionic acid compounds, and particularly relates to a method for measuring the accurate content of isomers in aryl propionic acid compounds by using an HPLC method.
Background
Loxoprofen sodium is an aryl propionic acid non-steroidal anti-inflammatory agent developed by the first Co-company of Japan, is a non-selective cyclooxygenase inhibitor, and achieves the purposes of anti-inflammatory and analgesic by inhibiting the synthesis of prostaglandins and unsaturated fatty acids catalyzed by arachidonic acid to generate drug effects. Loxoprofen is a prodrug of liver metabolism, which in vivo can bioconvert hydroxyl metabolites with 3 chiral centers, theoretically generating 8 stereoisomers, of which the 2's,1' r, 2's-trans hydroxyl active metabolites exert the main biological activity, i.e. compounds of formula i:
in the process of preparing the compounds, other 7 isomers which have no biological activity are inevitably generated. Only the method for detecting the content of loxoprofen sodium (JP 17) is described in the prior art, and the method can be used for detecting the content of loxoprofen sodium in finished products, but is not applicable to 8 isomers in loxoprofen active metabolites, and no reference method can be used at present.
In view of the above, a method which can effectively separate 8 isomers in loxoprofen active metabolite, is simple to operate, has easily available reagents, good reproducibility and little environmental pollution is developed and is very significant.
Disclosure of Invention
The invention aims to provide a method for detecting isomers in aryl propionic acid compounds.
The invention is mainly realized by the following technical scheme:
the invention provides a method for detecting isomers in aryl propionic acid compounds, which adopts high performance liquid chromatography detection, can detect 8 isomers including a compound of a formula I at the same time, and has a separation degree of not less than 1.5 between various chromatographic peaks;
according to an embodiment of the invention, the method chromatographic conditions are set as follows:
chromatographic column: the specification of the Chiralpak AD-H column is 4.6mm×250mm,5 μm;
mobile phase: a mixed solution of n-hexane, methanol, and isopropanol;
column temperature: 30-40 DEG C
Flow rate: 0.1-1.0 ml/min;
sample injection volume: 5-20 mul;
detection wavelength: 200-250 nm.
According to an embodiment of the present invention, the 8 isomers include a compound of formula i, impurity PY2, impurity PY3, impurity PY4, impurity PY5, impurity PY6, impurity PY7, impurity PY8, and the degree of separation between the respective chromatographic peaks is not less than 1.5;
according to an embodiment of the present invention, the method for detecting an isomer in an aryl propionic acid compound according to the present invention comprises the steps of:
(1) Sample preparation:
sample solution preparation: precisely measuring about 10mg of the product, placing in a 20ml measuring flask, diluting to scale with a diluent, shaking uniformly, and taking the product as a sample solution;
control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding diluent to dissolve and dilute to a scale, and shaking uniformly to obtain a control solution;
system applicability solution preparation: precisely measuring the compound reference substances of the formula I, PY2 reference substances, PY3 reference substances, PY4 reference substances, PY5 reference substances, PY6 reference substances, PY7 reference substances and PY8 reference substances, adding a diluent, shaking to dissolve and quantitatively dilute the compound reference substances to prepare a solution containing about 0.3mg of the compound reference substances of the formula I, about 25 mug of the PY2 reference substances, about 25 mug of the PY3 reference substances, about 25 mug of the PY4 reference substances, about 25 mug of the PY5 reference substances, about 25 mug of the PY6 reference substances, about 25 mug of the PY7 reference substances and about 25 mug of the PY8 reference substances in each 1ml of the solution as a system applicability solution;
(2) Setting chromatographic conditions:
chromatographic column: CHIRALPAKAD-H column with specification of 4.6mm×250mm,5 μm;
mobile phase: a mixed solution of n-hexane, methanol, and isopropanol;
column temperature: 30-40 DEG C
Flow rate: 0.1-1.0 ml/min;
sample injection volume: 5-20 mul;
detection wavelength: 200-250 nm;
(3) Detecting and calculating:
precisely measuring 20 mu 1 of system applicability solution, injecting into a liquid chromatograph, precisely measuring 20 mu 1 of control solution and 20 mu 1 of sample solution respectively, injecting into the liquid chromatograph, and recording chromatograms.
According to the embodiment of the invention, the diluent is a mixed solution of normal hexane, absolute ethyl alcohol and trifluoroacetic acid, and the volume ratio is 65-75:25-35:2, preferably 70:30:2.
According to the embodiment of the invention, the mobile phase is a mixed solution of n-hexane, methanol and isopropanol, and the volume ratio is 88: 8-10: 2 to 4.
According to an embodiment of the invention, the column temperature is 40 DEG C
According to an embodiment of the invention, the flow rate is 1.0ml/min.
According to an embodiment of the invention, the sample volume is 20 μl.
According to an embodiment of the invention, the wavelength is 220nm.
According to an embodiment of the present invention, the detection limit of the impurity is: examples of the chromatogram of the sample solution include those having retention times corresponding to the peaks of the impurities PY2, PY3, PY4, PY5, PY6, PY7 and PY8, wherein the peak area of PY5 is not more than 0.4 times, i.e., 2.0%, of the main peak area of the control solution, and the peak areas of the other impurities are not more than 0.2 times, i.e., 1.0%, of the main peak area of the control solution.
The invention has the beneficial effects that:
according to the method for detecting the isomers in the aryl propionic acid compounds, 8 isomers in the loxoprofen active metabolites can be completely separated, so that the quality of the loxoprofen active metabolites and the salt thereof can be effectively controlled, quality monitoring is effectively realized on the loxoprofen active metabolites and the salt products thereof, and the method has practical guiding significance for improving the quality of related products. The separation and measurement method has the advantages of strong specificity, high accuracy, simple and convenient operation, and wide popularization and application values, and can create wider economic benefits.
Drawings
FIG. 1 is a system applicability solution chromatogram measured under the chromatographic condition of comparative example 1 of the present invention.
FIG. 2 is a system applicability solution chromatogram measured under the chromatographic condition of comparative example 2 of the present invention.
FIG. 3 is a system applicability solution chromatogram measured under the condition of comparative example 3 chromatography of the present invention.
FIG. 4 is a system applicability solution chromatogram measured under the chromatographic condition of comparative example 4 of the present invention.
FIG. 5 is a system applicability solution chromatogram measured under the chromatographic conditions of example 1 of the present invention.
FIG. 6 is a system applicability solution chromatogram measured under the chromatographic conditions of example 2 of the present invention.
FIG. 7 is a system applicability solution chromatogram measured under the chromatographic conditions of example 3 of the present invention.
FIG. 8 is a system applicability solution chromatogram measured under the chromatographic conditions of example 4 of the present invention.
FIG. 9 is a linear diagram of the compound of formula I according to test example 2 of the present invention.
Detailed Description
The invention will now be described in further detail with reference to the following specific examples and figures, which are intended to be illustrative only and not limiting in any way, and the raw materials used, unless otherwise specified, may be either commercially available or self-made.
Patent CN106045842a discloses a process for preparing loxoprofen active metabolite, i.e. the compound of formula i. For the purpose of applicant's earlier research in published patents, the finished products of the compounds of formula I according to the present invention can be prepared by the above-mentioned methods. In the process of preparing the compounds, other 7 isomers which have no biological activity are inevitably generated. When the isomer analysis method is established, the reverse phase chromatography is firstly tried, the common liquid phase analysis method of the common reverse phase chromatographic column is adopted, the 8 isomers can not be completely separated, and then the normal phase chromatography is used for fumbling.
According to the invention, the optimal analysis conditions are optimized after a large number of experiments, chiral chromatographic columns CHIRALPAK AD-H are adopted, normal hexane, methanol and isopropanol are adopted as mobile phases, the column temperature is adjusted, the component proportion of the mobile phases is strictly controlled, the components of the diluent are adjusted, the separation degree between a main peak and other seven isomers is improved, the separation degree is more than 1.5, and the separation effect is outstanding. The isomer detection of the product mainly comprises the following optimization steps:
comparative example 1
The high performance liquid chromatography method for detecting the isomer in the aryl propionic acid compound specifically comprises the following steps:
(1) Chromatographic condition set (reversed phase chromatography + reversed phase C18 column):
chromatographic column: inertsuramin C18,3.0 mm. Times.100 mm,3 μm
Column temperature: 35 ℃;
flow rate: 0.4ml/min;
detection wavelength: 220nm;
sample injection amount: 20 μl;
mobile phase a:0.1% phosphoric acid in water, mobile phase B: acetonitrile;
gradient elution was performed as follows:
| t(min) | 0.01 | 30 | 50 | 50.1 | 80 |
| A(％) | 90 | 40 | 40 | 90 | 90 |
| B(％) | 10 | 60 | 60 | 10 | 10 |
(2) Preparing a diluent: methanol-water (30:70);
(3) Sample solution preparation: about 10mg of the product is taken and placed in a 20ml measuring flask, a proper amount of diluent is added, the mixture is shaken to dissolve, the diluent is diluted to a scale, and the mixture is shaken uniformly to be used as a sample solution;
(4) Control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain a control solution;
(5) System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
(6) And (3) detection: the precision measuring system applicability solution 20 mu 1 is injected into a liquid chromatograph, and the separation degree among the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 and the compound of the formula I is required to be in accordance with the requirements; precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatograms;
the test results are shown in FIG. 1, and only two chromatographic peaks are shown, wherein the peak outlet time is 26.019min and 26.549min respectively.
Comparative example 2
The high performance liquid chromatography method for detecting the isomer in the aryl propionic acid compound specifically comprises the following steps:
(1) Chromatographic condition setting (reversed phase chromatography + chiral column):
chromatographic column: CHIRALCEL OJ-RH,4.6 mm. Times.150 mm,5 μm
Column temperature: 35 ℃;
flow rate: 1.0ml/min;
detection wavelength: 220nm;
sample injection amount: 20 μl;
mobile phase a: pH3.5 aqueous solution (pH 3.5 adjusted with phosphoric acid), mobile phase B: acetonitrile;
gradient elution was performed as follows:
| t(min) | 0.01 | 30 | 40 | 41 | 50 |
| A(％) | 95 | 60 | 60 | 95 | 95 |
| B(％) | 5 | 40 | 40 | 5 | 5 |
(2) Preparing a diluent: methanol-water (30:70);
(3) Sample solution preparation: about 10mg of the product is taken and placed in a 20ml measuring flask, a proper amount of diluent is added, the mixture is shaken to dissolve, the diluent is diluted to a scale, and the mixture is shaken uniformly to be used as a sample solution;
(4) Control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain a control solution;
(5) System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
(6) And (3) detection: the precision measuring system applicability solution 20 mu 1 is injected into a liquid chromatograph, and the separation degree among the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 and the compound of the formula I is required to be in accordance with the requirements; precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatograms;
compared to comparative example 1, the chiral chromatographic column was replaced and the chromatographic conditions and gradient elution procedure were further adjusted. The test results are shown in FIG. 2, which shows 8 chromatographic peaks, but the degree of separation between the chromatographic peaks is poor.
Comparative example 3
The high performance liquid chromatography method for detecting the isomer in the aryl propionic acid compound specifically comprises the following steps:
(1) Chromatographic condition setting (normal phase chromatography + chiral column):
chromatographic column: CHIRALPAKAD-H,4.6 mm. Times.250 mm,5 μm
Column temperature: 30 ℃;
flow rate: 1.0ml/min;
detection wavelength: 220nm;
sample injection amount: 20 μl;
mobile phase a: n-heptane, mobile phase B: isopropyl alcohol;
gradient elution was performed as follows:
| t(min) | 0 | 20 | 40 | 40.1 | 50 |
| A(％) | 95 | 90 | 90 | 95 | 95 |
| B(％) | 5 | 10 | 10 | 5 | 5 |
(2) Preparing a diluent: absolute ethanol-n-hexane (30:70);
(3) Sample solution preparation: about 10mg of the product is taken and placed in a 20ml measuring flask, a proper amount of diluent is added, the mixture is shaken to dissolve, the diluent is diluted to a scale, and the mixture is shaken uniformly to be used as a sample solution;
(4) Control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain a control solution;
(5) System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
(6) And (3) detection: the precision measuring system applicability solution 20 mu 1 is injected into a liquid chromatograph, and the separation degree among the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 and the compound of the formula I is required to be in accordance with the requirements; precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatograms;
compared with comparative example 2, the chiral chromatographic column with different fillers is replaced, the chromatographic conditions and the gradient elution procedure are further adjusted, and the test result is shown in fig. 3, which shows 8 chromatographic peaks, and the separation degree between the peaks is obviously improved.
Comparative example 4
The high performance liquid chromatography method for detecting the isomer in the aryl propionic acid compound specifically comprises the following steps:
(1) Setting chromatographic conditions:
chromatographic column: CHIRALPAK AD-H,4.6 mm. Times.250 mm,5 μm
Column temperature: 30 ℃;
flow rate: 1.0ml/min;
detection wavelength: 220nm;
sample injection amount: 20 μl;
mobile phase a: n-heptane, mobile phase B: methanol, mobile phase C: isopropyl alcohol;
gradient elution was performed as follows:
| t(min) | 0 | 20 | 40 | 40.1 | 50 |
| A(％) | 90 | 85 | 85 | 90 | 90 |
| B(％) | 5 | 10 | 10 | 5 | 5 |
| C(％) | 5 | 5 | 5 | 5 | 5 |
(2) Preparing a diluent: absolute ethanol-n-hexane (30:70);
(3) Sample solution preparation: about 10mg of the product is taken and placed in a 20ml measuring flask, a proper amount of diluent is added, the mixture is shaken to dissolve, the diluent is diluted to a scale, and the mixture is shaken uniformly to be used as a sample solution;
(4) Control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain a control solution;
(5) System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
(6) And (3) detection: the precision measuring system applicability solution 20 mu 1 is injected into a liquid chromatograph, and the separation degree among the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 and the compound of the formula I is required to be in accordance with the requirements; precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatograms;
compared with comparative example 3, the mobile phase composition and gradient elution procedure were further adjusted, and the test results are shown in fig. 4, wherein the separation degree of only PY5 and PY3 is less than 1.5 (0.84), and the separation degree between other peaks is greater than 1.5.
Example 1
The high performance liquid chromatography method for detecting the isomer in the aryl propionic acid compound specifically comprises the following steps:
(1) Setting chromatographic conditions: (normal phase)
Chromatographic column: CHIRALPAKAD-H,4.6 mm. Times.250 mm,5 μm
Column temperature: 30 ℃;
flow rate: 1.0ml/min;
detection wavelength: 220nm;
sample injection amount: 20 μl;
the mobile phase was n-hexane-methanol-isopropanol (88:8:4);
(2) Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (70:30:2)
(3) Sample solution preparation: about 10mg of the product is taken, put into a 20ml measuring flask, added with a proper amount of diluent, shaken to dissolve, diluted to a scale with the diluent, and shaken uniformly to be used as a test solution.
(4) Control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain a control solution;
(5) System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
(6) And (3) detection: the precision measuring system applicability solution 20 mu 1 is injected into a liquid chromatograph, and the separation degree among the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 and the compound of the formula I is required to be in accordance with the requirements; precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatograms;
compared with comparative example 4, the mobile phase and the diluent are further adjusted, trifluoroacetic acid is added to the gradient elution and is changed to isocratic elution, the peak outlet time of the main peak and the impurity is shown in table 1, the test result is shown in fig. 5, the separation degree between all peaks is more than 1.5, and the impurities PY2, PY3, PY4, PY5, PY6, PY7 and PY8 are completely separated from the main component by the method.
TABLE 1
Example 2
The high performance liquid chromatography method for detecting the isomer in the aryl propionic acid compound specifically comprises the following steps:
(1) Setting chromatographic conditions:
chromatographic column: CHIRALPAKAD-H,4.6 mm. Times.250 mm,5 μm
Column temperature: 35 ℃;
flow rate: 1.0ml/min;
detection wavelength: 220nm;
sample injection amount: 20 μl;
the mobile phase was n-hexane-methanol-isopropanol (88:9:3);
(2) Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (70:30:2)
(3) Sample solution preparation: about 10mg of the product is taken, put into a 20ml measuring flask, added with a proper amount of diluent, shaken to dissolve, diluted to a scale with the diluent, and shaken uniformly to be used as a test solution.
(4) Control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain a control solution;
(5) System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
(6) And (3) detection: the precision measuring system applicability solution 20 mu 1 is injected into a liquid chromatograph, and the separation degree among the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 and the compound of the formula I is required to be in accordance with the requirements; precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatograms;
in comparison with example 1, the column temperature and the mobile phase ratio were further adjusted, the peak-out times of the main peak and the impurity were shown in table 2, the test results were shown in fig. 6, the degree of separation between all peaks was greater than 1.5, and the impurities PY2, PY3, PY4, PY5, PY6, PY7, and PY8 were completely separated from the main component by this method.
TABLE 2
Example 3
The high performance liquid chromatography method for detecting the isomer in the aryl propionic acid compound specifically comprises the following steps:
(1) Setting chromatographic conditions: (normal phase)
Chromatographic column: CHIRALPAKAD-H,4.6 mm. Times.250 mm,5 μm
Column temperature: 40 ℃;
flow rate: 1.0ml/min;
detection wavelength: 220nm;
sample injection amount: 20 μl;
the mobile phase was n-hexane-methanol-isopropanol (88:10:2);
(2) Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (65:35:2)
(3) Sample solution preparation: about 10mg of the product is taken, put into a 20ml measuring flask, added with a proper amount of diluent, shaken to dissolve, diluted to a scale with the diluent, and shaken uniformly to be used as a test solution.
(4) Control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain a control solution;
(5) System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
(6) And (3) detection: the precision measuring system applicability solution 20 mu 1 is injected into a liquid chromatograph, and the separation degree among the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 and the compound of the formula I is required to be in accordance with the requirements; precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatograms;
compared with example 2, the column temperature, mobile phase proportion and diluent proportion are further adjusted, the peak outlet time of the main peak and the impurities is shown in table 3, the test result is shown in fig. 7, the separation degree between all peaks is more than 1.5, and the impurities PY2, PY3, PY4, PY5, PY6, PY7 and PY8 are completely separated from the main component by the method.
TABLE 3 Table 3
Example 4
The high performance liquid chromatography method for detecting the isomer in the aryl propionic acid compound specifically comprises the following steps:
(1) Setting chromatographic conditions: (normal phase)
Chromatographic column: CHIRALPAKAD-H,4.6 mm. Times.250 mm,5 μm
Column temperature: 40 ℃;
flow rate: 1.0ml/min;
detection wavelength: 220nm;
sample injection amount: 20 μl;
the mobile phase was n-hexane-methanol-isopropanol (88:8:4);
(2) Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (75:25:2)
(3) Sample solution preparation: about 10mg of the product is taken, put into a 20ml measuring flask, added with a proper amount of diluent, shaken to dissolve, diluted to a scale with the diluent, and shaken uniformly to be used as a test solution.
(4) Control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain a control solution;
(5) System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
(6) And (3) detection: the precision measuring system applicability solution 20 mu 1 is injected into a liquid chromatograph, and the separation degree among the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 and the compound of the formula I is required to be in accordance with the requirements; precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatograms;
in comparison with example 3, the column temperature and the diluent ratio were further adjusted, the peak-out times of the main peak and the impurity are shown in table 4, the test results are shown in fig. 8, the degree of separation between all peaks is greater than 1.5, and the impurities PY2, PY3, PY4, PY5, PY6, PY7, PY8 were completely separated from the main component by this method.
TABLE 4 Table 4
Test example 1 specificity
Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (70:30:2)
System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
And respectively injecting the diluent and the system applicability solution into a liquid chromatograph, and examining whether a blank solvent (diluent) has interference on main peaks and various isomers and separating the isomers from the main peaks.
Test example 2 linearity and Range
Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (70:30:2)
A main component stock solution: a compound of formula I is taken as a reference substance, a proper amount of the reference substance is precisely weighed, a diluting agent is added, and the reference substance is shaken to be dissolved and quantitatively diluted to prepare a solution containing about 0.5mg of the compound of formula I in 1ml as a stock solution.
Linear solution: and diluting the main component stock solution with diluent to obtain solutions with different concentrations (quantitative limit, 20%, 30%, 40%, 100%, 200%).
The linear solutions with different concentrations are respectively taken and injected into a liquid chromatograph, and the linear relation between the concentration and the peak response is examined, as shown in figure 9.
Test example 3 detection limit and quantitative limit
Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (70:30:2)
A main component stock solution: a compound of formula I is taken as a reference substance, a proper amount of the reference substance is precisely weighed, a diluting agent is added, and the reference substance is shaken to be dissolved and quantitatively diluted to prepare a solution containing about 0.5mg of the compound of formula I in 1ml as a stock solution.
LOQ solution: and respectively taking a proper amount of each impurity stock solution and a proper amount of the main component stock solution, and diluting the stock solution with a diluent to prepare a solution with a certain concentration until the signal to noise ratio of each impurity peak and main peak is more than or equal to 10.
LOD solution: and respectively taking a proper amount of each impurity stock solution and a proper amount of the main component stock solution, and diluting the stock solution with a diluent to prepare a solution with a certain concentration until the signal to noise ratio of each impurity peak and main peak is more than or equal to 3.
Test example 4 precision
Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (70:30:2)
System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
Sample solution preparation: about 10mg of the product is taken, put into a 20ml measuring flask, added with a proper amount of diluent, shaken to dissolve, diluted to a scale with the diluent, and shaken uniformly to be used as a test solution. Six portions were prepared in parallel.
Control solution preparation: precisely measuring 1ml of the sample solution, placing into a 20ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly.
The solutions were injected into a liquid chromatograph, respectively, and the reproducibility of the method was examined.
Test example 5 solution stability
And (3) preparing the solution with the same precision as in test example 4, and respectively testing the stability of the sample solution and the control solution by using a liquid chromatograph at different room temperature.
Test example 6 durability
Preparing a diluent: n-hexane-absolute ethanol-trifluoroacetic acid (70:30:2)
System applicability solution preparation: taking a proper amount of each of the compound of formula I, PY2, PY3, PY4, PY5, PY6, PY7 and PY8, precisely weighing, adding a diluent, shaking to dissolve and quantitatively dilute the compound of formula I into a solution containing about 0.3mg of each of the compound of formula I, the PY2, PY3, PY4, PY5, PY6, PY7 and PY8 in 1ml, and taking the solution as a system applicability solution.
The separation of each isomer and each isomer from the main peak was examined by taking the system-applicable solutions and slightly varying the flow rate, column temperature and organic phase ratio of the chromatographic conditions.
The detection method is verified in terms of specificity, linearity and range, detection limit and quantitative limit, precision, solution stability, durability and the like, and the verification result is shown in table 5.
TABLE 5
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Claims (10)
1. A method for detecting isomers in aryl propionic acid compounds is characterized in that the method adopts high performance liquid chromatography detection, 8 isomers comprising a compound of a formula I can be detected simultaneously, and the separation degree between each chromatographic peak is not less than 1.5;
the chromatographic conditions of the detection method are set as follows:
chromatographic column: the ChiralpakAD-H column has the specification of 4.6mm multiplied by 250mm and 5 mu m;
mobile phase: a mixed solution of n-hexane, methanol, and isopropanol;
column temperature: 30-40 DEG C
Flow rate: 0.1-1.0 ml/min;
sample injection volume: 5-20 mul;
detection wavelength: 200-250 nm.
2. The method of claim 1, wherein the 8 isomers comprise a compound of formula i, impurity PY2, impurity PY3, impurity PY4, impurity PY5, impurity PY6, impurity PY7, impurity PY8, the degree of separation between the respective chromatographic peaks being no less than 1.5;
3. the method according to claim 1, comprising the steps of:
(1) Sample preparation:
sample solution preparation: precisely measuring about 10mg of the product, placing in a 20ml measuring flask, diluting to scale with a diluent, shaking uniformly, and taking the product as a sample solution;
control solution preparation: precisely measuring 1ml of the sample solution, placing the sample solution into a 20ml measuring flask, adding diluent to dissolve and dilute to a scale, and shaking uniformly to obtain a control solution;
system applicability solution preparation: precisely measuring the compound reference substance of the formula I, the impurity PY2 reference substance, the PY3 reference substance, the PY4 reference substance, the PY5 reference substance, the PY6 reference substance, the PY7 reference substance and the PY8 reference substance in proper amounts, adding a diluting agent, shaking to dissolve and quantitatively dilute the compound reference substance of the formula I to prepare a solution with about 0.3mg of the compound reference substance of the formula I, the PY2 reference substance, the PY3 reference substance, the PY4 reference substance, the PY5 reference substance, the PY6 reference substance, the PY7 reference substance and the PY8 reference substance in each 1ml, and taking the solution as a system applicability solution;
(2) Setting chromatographic conditions:
chromatographic column: CHIRALPAKAD-H column with specification of 4.6mm×250mm,5 μm;
mobile phase: a mixed solution of n-hexane, methanol, and isopropanol;
column temperature: 30-40 DEG C
Flow rate: 0.1-1.0 ml/min;
sample injection volume: 5-20 mul;
detection wavelength: 200-250 nm;
(3) Detecting and calculating:
precisely measuring 20 mu 1 of system applicability solution, injecting into a liquid chromatograph, precisely measuring 10 mu 1 of control solution and 10 mu 1 of test solution respectively, injecting into the liquid chromatograph, and recording chromatograms.
4. The method according to claim 3, wherein the diluent is a mixed solution of n-hexane, absolute ethyl alcohol and trifluoroacetic acid, and the volume ratio is 65-75:25-35:2; preferably 70:30:2.
5. A method according to claim 3, wherein the mobile phase is a mixed solution of n-hexane, methanol and isopropanol in a volume ratio of 88: 8-10: 2 to 4.
6. A method according to claim 3, wherein the column temperature is 40 ℃.
7. A method according to claim 3, wherein the flow rate is 1.0ml/min.
8. A method according to claim 3, wherein the sample volume is 20 μl.
9. A method according to claim 3, wherein the wavelength is 220nm.
10. A method according to claim 3, wherein the detection limit of the impurity is: examples of the chromatogram of the sample solution include those having retention times corresponding to the peaks of the impurities PY2, PY3, PY4, PY5, PY6, PY7 and PY8, wherein the peak area of PY5 is not more than 0.4 times, i.e., 2.0%, of the main peak area of the control solution, and the peak areas of the other impurities are not more than 0.2 times, i.e., 1.0%, of the main peak area of the control solution.
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