CN110568100B - Mitiglinide calcium R-isomer detection method - Google Patents
Mitiglinide calcium R-isomer detection method Download PDFInfo
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- CN110568100B CN110568100B CN201910862258.5A CN201910862258A CN110568100B CN 110568100 B CN110568100 B CN 110568100B CN 201910862258 A CN201910862258 A CN 201910862258A CN 110568100 B CN110568100 B CN 110568100B
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- mitiglinide calcium
- isomer
- mitiglinide
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- 229960003365 mitiglinide Drugs 0.000 title claims abstract description 103
- PMRVFZXOCRHXFE-FMEJWYFOSA-L Kad 1229 Chemical compound [Ca+2].C([C@@H](CC(=O)N1C[C@@H]2CCCC[C@@H]2C1)C(=O)[O-])C1=CC=CC=C1.C([C@@H](CC(=O)N1C[C@@H]2CCCC[C@@H]2C1)C(=O)[O-])C1=CC=CC=C1 PMRVFZXOCRHXFE-FMEJWYFOSA-L 0.000 title claims abstract 29
- 238000001514 detection method Methods 0.000 title claims description 14
- 239000000243 solution Substances 0.000 claims abstract description 53
- 238000002360 preparation method Methods 0.000 claims abstract description 40
- 238000005303 weighing Methods 0.000 claims abstract description 34
- 239000013558 reference substance Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000012085 test solution Substances 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 11
- 239000012088 reference solution Substances 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 8
- 238000002347 injection Methods 0.000 claims abstract description 5
- 239000007924 injection Substances 0.000 claims abstract description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 3
- 238000010606 normalization Methods 0.000 claims abstract description 3
- WPGGHFDDFPHPOB-BBWFWOEESA-N mitiglinide Chemical compound C([C@@H](CC(=O)N1C[C@@H]2CCCC[C@@H]2C1)C(=O)O)C1=CC=CC=C1 WPGGHFDDFPHPOB-BBWFWOEESA-N 0.000 claims description 10
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 claims description 9
- QEVLNUAVAONTEW-UZYHXJQGSA-L calcium;(2s)-4-[(3as,7ar)-1,3,3a,4,5,6,7,7a-octahydroisoindol-2-yl]-2-benzyl-4-oxobutanoate;dihydrate Chemical compound O.O.[Ca+2].C([C@@H](CC(=O)N1C[C@@H]2CCCC[C@@H]2C1)C(=O)[O-])C1=CC=CC=C1.C([C@@H](CC(=O)N1C[C@@H]2CCCC[C@@H]2C1)C(=O)[O-])C1=CC=CC=C1 QEVLNUAVAONTEW-UZYHXJQGSA-L 0.000 description 64
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 14
- 239000000523 sample Substances 0.000 description 11
- 238000000926 separation method Methods 0.000 description 10
- 238000012216 screening Methods 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 239000012467 final product Substances 0.000 description 7
- 239000011259 mixed solution Substances 0.000 description 7
- 230000003044 adaptive effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 4
- GTOFKXZQQDSVFH-VIFPVBQESA-N (r)-2-benzylsuccinate Chemical group OC(=O)C[C@@H](C(O)=O)CC1=CC=CC=C1 GTOFKXZQQDSVFH-VIFPVBQESA-N 0.000 description 3
- KYILORDWJFEQBS-UHFFFAOYSA-N 2-benzylidenebutanedioic acid Chemical compound OC(=O)CC(C(O)=O)=CC1=CC=CC=C1 KYILORDWJFEQBS-UHFFFAOYSA-N 0.000 description 2
- GTOFKXZQQDSVFH-UHFFFAOYSA-N 2-benzylsuccinic acid Chemical compound OC(=O)CC(C(O)=O)CC1=CC=CC=C1 GTOFKXZQQDSVFH-UHFFFAOYSA-N 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000003914 insulin secretion Effects 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- ODSNARDHJFFSRH-OCAPTIKFSA-N (3as,7ar)-2,3,3a,4,5,6,7,7a-octahydro-1h-isoindole Chemical compound C1CCC[C@@H]2CNC[C@@H]21 ODSNARDHJFFSRH-OCAPTIKFSA-N 0.000 description 1
- GTOFKXZQQDSVFH-SECBINFHSA-N (R)-2-benzylsuccinic acid Chemical compound OC(=O)C[C@H](C(O)=O)CC1=CC=CC=C1 GTOFKXZQQDSVFH-SECBINFHSA-N 0.000 description 1
- WVXPTHZHWXOLNK-UHFFFAOYSA-N 4-(2,3-dihydro-1h-isoindol-1-yl)butanoic acid Chemical compound C1=CC=C2C(CCCC(=O)O)NCC2=C1 WVXPTHZHWXOLNK-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- ARLZGEXVMUDUQZ-UHFFFAOYSA-N O.O.[Ca] Chemical compound O.O.[Ca] ARLZGEXVMUDUQZ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000009460 calcium influx Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000003112 degranulating effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000009207 exercise therapy Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229940126904 hypoglycaemic agent Drugs 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- -1 mitiglinide benzyl ester Chemical class 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Classifications
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- 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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
Abstract
The invention relates to a method for detecting an R-isomer of mitiglinide calcium, which adopts a high performance liquid chromatography and comprises the following steps: 1) preparing a test solution: taking a proper amount of mitiglinide calcium raw material test products, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking uniformly to obtain the mitiglinide calcium injection; 2) preparation of mitiglinide calcium control solution: taking a proper amount of mitiglinide calcium reference substance, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg of mitiglinide calcium per 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation; 3) preparation of mitiglinide calcium R-isomer reference solution: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg of mitiglinide calcium R-isomer in each 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation; 4) the determination method comprises the following steps: and (3) respectively injecting 20 mu l of each solution obtained in the step 1), the step 2) and the step 3) into a high performance liquid chromatograph to obtain a chromatogram, and calculating the content of the R-isomer of the mitiglinide calcium in the mitiglinide calcium raw material drug according to a normalization method according to the peak area in the chromatogram.
Description
Technical Field
The invention relates to an identification method of mitiglinide calcium R-isomer.
Background
Mitiglinide calcium dihydrate, the chemical name of which is: bis [ (2s) -2-benzyl-3- (cis-hexahydroisoindol-2-carbonyl) propionic acid ] monocalcium dihydrate, having the following structural formula:
mitiglinide calcium is a hypoglycemic agent developed by Nippon orange company, and is a novel benzylsuccinic acid insulin secretion promoter. Mitiglinide stimulates insulin secretion by closing ATP-dependent K + channels on the pancreatic beta cell membrane, causing Ca2+ influx, increasing intracellular Ca2+ concentration and degranulating extracellular insulin-containing vesicles. Mitiglinide calcium tablet is used for controlling postprandial blood sugar of type 2 diabetes patients who cannot achieve satisfactory effects through food therapy and exercise therapy. The difference between mitiglinide calcium and its chiral isomer exists only in the position of S-benzylsuccinic acid fragment in the structure, and R-benzylsuccinic acid is contained in S-benzylsuccinic acid, and is called as: the R-isomer of mitiglinide calcium has the following structural formula:
the R-isomer of mitiglinide calcium is used as an impurity component in the mitiglinide calcium bulk drug and must be controlled within a certain limit, so the content of the R-isomer of mitiglinide calcium in the mitiglinide calcium bulk drug needs to be detected and analyzed, and the existing standard discloses the following detection method:
First, national drug Standard YBH00192012
Second, national drug Standard YBH04122012
The R-isomer detection method can separate the mitiglinide calcium peak from the R-isomer peak, but the separation degree cannot meet the legal requirement.
See fig. 6, the chromatographic conditions are as follows:
chromatographic conditions and system applicability test:
and (3) chromatographic column: chiral chromatographic column (Sumichiral OA-3300250X 4.6mm 5 μm);
mobile phase: acetonitrile (80:20) at 0.03mol/L ammonium acetate in methanol;
detection wavelength: 210 nm;
flow rate: 0.8ml/min
Column temperature: 30 deg.C
Preparation of a test solution: taking a proper amount of the sample, precisely weighing, adding mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking up to obtain the final product.
Preparation of control solution: precisely measuring 1ml of the sample, placing into a 100ml volumetric flask, adding the mobile phase to dilute to a scale, and shaking up to obtain the product.
Preparing system adaptability: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding methanol to prepare a solution containing 0.2mg per 1ml, and shaking up to obtain the mitiglinide calcium preparation.
The determination method comprises injecting 20 μ l of system adaptive solution into a liquid chromatograph, sequentially injecting mitiglinide calcium and R isomer in peak order, and injecting 20 μ l of test solution and control solution into the chromatograph, wherein the number of theoretical plates is not less than 2000 calculated according to mitiglinide peak.
Disclosure of Invention
On the basis of the prior art, the invention improves the chromatographic conditions and the preparation of the test solution, and the detection effect is superior to that of the prior art.
The invention provides a method for measuring the content of R-isomer of mitiglinide calcium in mitiglinide calcium raw material medicine, which adopts a high performance liquid chromatography and comprises the following steps:
1) preparation of a test solution: taking a proper amount of mitiglinide calcium raw material test products, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking uniformly to obtain the mitiglinide calcium injection.
2) Preparation of mitiglinide calcium reference solution: taking a proper amount of mitiglinide calcium reference substance, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg of mitiglinide calcium per 1ml, and shaking up to obtain the mitiglinide calcium injection.
3) Preparation of mitiglinide calcium R-isomer reference solution: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg of mitiglinide calcium R-isomer per 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation.
4) The determination method comprises the following steps: and (3) respectively injecting 20 mu l of each solution obtained in the step 1), the step 2) and the step 3) into a high performance liquid chromatograph to obtain a chromatogram, and calculating the content of the R-isomer of the mitiglinide calcium in the mitiglinide calcium raw material drug according to a normalization method according to the peak area in the chromatogram.
Wherein, the peak sequences of the components in the chromatogram are mitiglinide calcium and mitiglinide calcium R isomers in turn, and the number of theoretical plates is not less than 2000 calculated according to mitiglinide peaks.
Wherein the chromatographic conditions of the high performance liquid chromatograph are as follows:
a chromatographic column: chiral chromatographic column Ultimate Amy-DR (250X 4.6mm 5 μm) is adopted;
mobile phase: acetonitrile-trifluoroacetic acid (100: 0.05-0.2);
detection wavelength: 210 nm.
Flow rate: 0.3-0.8ml/min
The mitiglinide calcium raw material medicine is prepared according to the method in the prior art, and the method comprises the following steps:
benzylidene succinic acid synthesis → benzylsuccinic acid synthesis → (S) -benzylsuccinic acid- (R) -a-phenethylamine salt synthesis → (S) -benzylsuccinic acid synthesis → cis-hexahydroisoindoline synthesis → isoindoline butyric acid synthesis → mitiglinide benzyl ester synthesis → 2S-isoindoline butyric acid synthesis → mitiglinide calcium.
The method is established by screening, and the screening process comprises the following steps:
1. wavelength screening: scanning the mitiglinide calcium reference substance solution and the R-isomer reference substance mixed solution in a 200-400nm ultraviolet spectrophotometer to determine that the maximum absorption is generated at 210nm without interference.
2. Screening of mobile phase:
the sample treatment method comprises the following steps: taking a proper amount of mitiglinide calcium and an R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a mixed solution containing 0.2mg of mitiglinide calcium and the R-isomer per 1ml, and shaking uniformly to obtain the mitiglinide.
The following different mobile phases were used as sample injections, in acetonitrile: the ratio of trifluoroacetic acid is preferably 100: 0.1.
| Diluent | Ratio of | Degree of separation | Number of theoretical plates |
| Acetonitrile: trifluoroacetic acid | 100:0.05 | 1.50 | 2134 |
| Acetonitrile: trifluoroacetic acid | 100:0.1 | 2.63 | 2530 |
| Acetonitrile: trifluoroacetic acid | 100:0.2 | 2.01 | 2076 |
| Methanol: trifluoroacetic acid | 100:0.05 | 1.25 | 1965 |
| Methanol: trifluoroacetic acid | 100:0.1 | 1.10 | 1852 |
| Methanol: trifluoroacetic acid | 100:0.2 | 0.93 | 1869 |
3. System applicability
And (4) screening of chromatographic columns, namely selecting different chromatographic columns for experiment, and optimizing the resolution of chiral chromatographic column Ultimate Amy-DR.
| Chromatographic column | Bonded phase | Degree of separation |
| Chiral chromatographic column | Ultimate Amy-SR | 2.37 |
| Chiral chromatographic column | Ultimate Amy-DR | 2.65 |
| Chiral chromatographic column | Sumichiral OA-3300 | 0.70 |
| Chiral chromatographic column | Sumichiral OA-3100 | 1.03 |
4. Specificity
Preparation of a test solution: taking a proper amount of the sample, precisely weighing, adding mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking up to obtain the final product.
Preparation of negative sample solution: and (4) taking a blank solvent, and filtering to obtain the product.
Preparation of control solutions: taking a proper amount of R-isomer reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Injecting 20 μ l of each of the above 3 solutions into a liquid chromatograph, and recording chromatogram.
As a result: the mitiglinide calcium in the test solution and the R-isomer in the reference solution reach baseline separation. The negative sample solution has no interference, and the product has good specificity.
See figures 1-5.
5. Durability
5.1 mobile phase: acetonitrile-trifluoroacetic acid (100: 0.1);
The theoretical plate numbers of 5.2 flow rates of 0.3ml/min, 0.5ml/min and 0.8ml/min were not less than 2000, but the degree of separation was most preferably 0.5 ml/min.
| Flow rate of flow | Number of theoretical plate | Degree of separation |
| 0.3ml/min | 2170 | 2.45 |
| 0.5ml/min | 2480 | 2.63 |
| 0.8ml/min | 2010 | 2.60 |
5.3, different instruments are adopted for detection, the method has good stability, no tailing of peaks, and the separation degree of more than 1.5.
| Instrument for measuring the position of a moving object | Model number | Degree of separation |
| Agilent high performance liquid chromatograph | 1260 | 2.60 |
| Waters high performance liquid chromatograph | 2695 | 2.51 |
The comparative data of the present invention and the prior art are as follows:
| type (B) | Degree of separation | Number of theoretical plate |
| The invention | 2.63 | 2639 |
| National drug Standard YBH00192012 | 0.76 | 2315 |
| National drug Standard YBH04122012 | 1.45 | 2473 |
The core technical scheme of the invention is as follows:
1. mobile phase: the mobile phase acetonitrile-trifluoroacetic acid (100: 0.1) was optimized by screening for 6 mobile phase ratios.
2. Flow rate: the flow rate of 0.5ml/min is optimal by screening at 3 flow rates.
3. A chromatographic column: the chiral chromatographic column Ultimate Amy-DR is optimized by screening different manufacturers and different bonding phases.
The invention has the following advantages:
provides the screening of the chromatographic column to successfully solve the problem of separating mitiglinide calcium from the isomers thereof.
Description of the drawings:
FIG. 1 negative sample
FIG. 2 is a drawing: r-isomer control
FIG. 3 is a drawing: mitiglinide calcium reference substance
FIG. 4 is a drawing: system adaptability
FIG. 5: test article
FIG. 6: system adaptability (old method)
The specific implementation mode is as follows:
the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.
Example 1:
chromatographic conditions and system applicability test:
a chromatographic column: chiral chromatographic column Ultimate Amy-DR (250X 4.6mm 5 μm) is adopted;
mobile phase: acetonitrile-trifluoroacetic acid (100: 0.1);
detection wavelength: 210 nm.
Flow rate: 0.3ml/min
Column temperature: 30 deg.C
Preparation of a test solution: taking a proper amount of the sample, precisely weighing, adding mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking up to obtain the final product.
Preparation of mitiglinide calcium reference solution: taking appropriate amount of reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparation of R-isomer control solution: taking a proper amount of R-isomer reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparing system adaptability: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a mixed solution containing 0.2mg of mitiglinide calcium R-isomer in each 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation.
The determination method comprises injecting 20 μ l of system adaptive solution into a liquid chromatograph, sequentially injecting Mitiglinide calcium and R isomer in peak order, and injecting 20 μ l of test solution and control solution into the chromatograph, wherein the number of theoretical plates is not less than 2000 calculated according to Mitiglinide peak.
Example 2:
chromatographic conditions and system applicability test:
a chromatographic column: chiral chromatographic column Ultimate Amy-DR (250X 4.6mm 5 μm) is adopted;
mobile phase: acetonitrile-trifluoroacetic acid (100: 0.1);
detection wavelength: 210 nm.
Flow rate: 0.5ml/min
Column temperature: 30 deg.C
Preparation of a test solution: taking a proper amount of the sample, precisely weighing, adding mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking up to obtain the final product.
Preparation of mitiglinide calcium reference solution: taking appropriate amount of reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparation of R-isomer control solution: taking a proper amount of R-isomer reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparing system adaptability: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a mixed solution containing 0.2mg of mitiglinide calcium R-isomer in each 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation.
The determination method comprises injecting 20 μ l of system adaptive solution into a liquid chromatograph, sequentially injecting mitiglinide calcium and R isomer in peak order, and injecting 20 μ l of test solution and control solution into the chromatograph, wherein the number of theoretical plates is not less than 2000 calculated according to mitiglinide peak.
Example 3:
chromatographic conditions and system applicability test:
a chromatographic column: chiral chromatographic column Ultimate Amy-DR (250X 4.6mm 5 μm) is adopted;
mobile phase: acetonitrile-trifluoroacetic acid (100: 0.1);
detection wavelength: 210 nm.
Flow rate: 0.8ml/min
Column temperature: 30 deg.C
Preparation of a test solution: taking a proper amount of the sample, precisely weighing, adding mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking up to obtain the final product.
Preparation of mitiglinide calcium reference solution: taking appropriate amount of reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparation of R-isomer control solution: taking a proper amount of R-isomer reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparing system adaptability: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a mixed solution containing 0.2mg of mitiglinide calcium R-isomer in each 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation.
The determination method comprises injecting 20 μ l of system adaptive solution into a liquid chromatograph, sequentially injecting mitiglinide calcium and R isomer in peak order, and injecting 20 μ l of test solution and control solution into the chromatograph, wherein the number of theoretical plates is not less than 2000 calculated according to mitiglinide peak.
Example 4:
chromatographic conditions and system applicability test:
and (3) chromatographic column: chiral chromatographic column Ultimate Amy-DR (250X 4.6mm 5 μm) is adopted;
mobile phase: acetonitrile-trifluoroacetic acid (100: 0.2);
detection wavelength: 210 nm.
Flow rate: 0.5ml/min
Column temperature: 30 deg.C
Preparation of a test solution: taking a proper amount of the sample, precisely weighing, adding mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking up to obtain the final product.
Preparation of mitiglinide calcium reference solution: taking appropriate amount of reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparation of R-isomer control solution: taking a proper amount of R-isomer reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparing system adaptability: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a mixed solution containing 0.2mg of mitiglinide calcium R-isomer in each 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation.
The determination method comprises injecting 20 μ l of system adaptive solution into a liquid chromatograph, sequentially injecting mitiglinide calcium and R isomer in peak order, and injecting 20 μ l of test solution and control solution into the chromatograph, wherein the number of theoretical plates is not less than 2000 calculated according to mitiglinide peak.
Example 5
Chromatographic conditions and system applicability test:
a chromatographic column: chiral chromatographic column Ultimate Amy-DR (250X 4.6mm 5 μm) is adopted;
mobile phase: acetonitrile-trifluoroacetic acid (100: 0.05);
detection wavelength: 210 nm.
Flow rate: 0.5ml/min
Column temperature: 30 deg.C
Preparation of a test solution: taking a proper amount of the sample, precisely weighing, adding mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking up to obtain the final product.
Preparation of mitiglinide calcium reference solution: taking appropriate amount of reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparation of R-isomer control solution: taking a proper amount of R-isomer reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.2mg per 1ml, and shaking.
Preparing system adaptability: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a mixed solution containing 0.2mg of mitiglinide calcium R-isomer in each 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation.
The determination method comprises injecting 20 μ l of system adaptive solution into a liquid chromatograph, sequentially injecting Mitiglinide calcium and R isomer in peak order, and injecting 20 μ l of test solution and control solution into the chromatograph, wherein the number of theoretical plates is not less than 2000 calculated according to Mitiglinide peak.
The results of comparing the above 5 examples are as follows:
| types of | Degree of separation | Number of theoretical plate |
| Example 1 | 2.30 | 2159 |
| Example 2 | 2.69 | 2487 |
| Example 3 | 2.53 | 2031 |
| Example 4 | 2.09 | 2028 |
| Example 5 | 1.69 | 2003 |
Claims (1)
1. A method for detecting R-isomer of mitiglinide calcium by adopting high performance liquid chromatography comprises the following steps:
1) preparation of a test solution: taking a proper amount of mitiglinide calcium raw material test products, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg per 1ml, and shaking uniformly to obtain the mitiglinide calcium injection;
2) preparation of mitiglinide calcium reference solution: taking a proper amount of mitiglinide calcium reference substance, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg of mitiglinide calcium per 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation;
3) preparation of mitiglinide calcium R-isomer reference solution: taking a proper amount of mitiglinide calcium R-isomer reference substance, precisely weighing, adding a mobile phase to prepare a solution containing 0.2mg of mitiglinide calcium R-isomer in each 1ml, and shaking uniformly to obtain the mitiglinide calcium preparation;
4) the determination method comprises the following steps: respectively injecting 20 mu l of each solution obtained in the step 1), the step 2) and the step 3) into a high performance liquid chromatograph to obtain a chromatogram, and calculating the content of R-isomer of mitiglinide calcium in mitiglinide calcium raw material drug according to a peak area in the chromatogram by a normalization method;
wherein, the peak sequences of the components in the chromatogram are mitiglinide calcium and mitiglinide calcium R isomers in turn, and the number of theoretical plates is not less than 2000 calculated according to mitiglinide peaks;
Wherein the chromatographic conditions of the high performance liquid chromatograph are as follows:
and (3) chromatographic column: a chiral chromatographic column Ultimate Amy-DR with the model of 250 multiplied by 4.6mm and 5 mu m is adopted;
mobile phase: with acetonitrile-trifluoroacetic acid ═ 100: 0.1;
detection wavelength: 210 nm;
flow rate: 0.5 ml/min.
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