CN114184728A - Methylprednisolone intermediate analysis method - Google Patents
Methylprednisolone intermediate analysis method Download PDFInfo
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- CN114184728A CN114184728A CN202111460724.0A CN202111460724A CN114184728A CN 114184728 A CN114184728 A CN 114184728A CN 202111460724 A CN202111460724 A CN 202111460724A CN 114184728 A CN114184728 A CN 114184728A
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- methylprednisolone
- methanol
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- VHRSUDSXCMQTMA-PJHHCJLFSA-N 6alpha-methylprednisolone Chemical compound C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)CO)CC[C@H]21 VHRSUDSXCMQTMA-PJHHCJLFSA-N 0.000 title claims abstract description 69
- 229960004584 methylprednisolone Drugs 0.000 title claims abstract description 67
- 238000004458 analytical method Methods 0.000 title claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 105
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 26
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000000126 substance Substances 0.000 claims abstract description 15
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 21
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 15
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- 239000011630 iodine Substances 0.000 claims description 9
- 229910052740 iodine Inorganic materials 0.000 claims description 9
- 150000007524 organic acids Chemical class 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- 239000012491 analyte Substances 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000006192 iodination reaction Methods 0.000 claims description 6
- 230000026045 iodination Effects 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- BRPQOXSCLDDYGP-UHFFFAOYSA-N calcium oxide Chemical compound [O-2].[Ca+2] BRPQOXSCLDDYGP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000292 calcium oxide Substances 0.000 claims description 3
- ODINCKMPIJJUCX-UHFFFAOYSA-N calcium oxide Inorganic materials [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 claims description 3
- 238000006356 dehydrogenation reaction Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims 2
- 238000000926 separation method Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 description 11
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 230000014759 maintenance of location Effects 0.000 description 9
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 239000000543 intermediate Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000003862 glucocorticoid Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000003266 anti-allergic effect Effects 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 description 1
- 208000005676 Adrenogenital syndrome Diseases 0.000 description 1
- 208000027932 Collagen disease Diseases 0.000 description 1
- 208000008448 Congenital adrenal hyperplasia Diseases 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940124645 emergency medicine Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000030533 eye disease Diseases 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 229960001293 methylprednisolone acetate Drugs 0.000 description 1
- PLBHSZGDDKCEHR-LFYFAGGJSA-N methylprednisolone acetate Chemical compound C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)COC(C)=O)CC[C@H]21 PLBHSZGDDKCEHR-LFYFAGGJSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 201000006489 suppurative thyroiditis Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Steroid Compounds (AREA)
Abstract
The invention discloses an analysis method of a methylprednisolone intermediate, which comprises the steps of adopting a high performance liquid chromatography to elute a substance to be detected; the chromatographic conditions are as follows: the stationary phase is a chromatographic column of octadecylsilane chemically bonded silica, and the mobile phase A is water and the mobile phase B is methanol in the mobile phase; the volume ratio of the water to the methanol is 50:50-67: 33; the substance to be detected comprises methylprednisolone monoiodide and/or methylprednisolone diiodide. The analysis method provided by the invention has the characteristics of good separation degree, simplicity, rapidness, strong specificity, high sensitivity and the like.
Description
Technical Field
The invention relates to the field of medicine detection and analysis, in particular to a detection and analysis method of a methylprednisolone intermediate.
Background
Methylprednisolone is 11 beta, 17, 21-trihydroxy-6 alpha-methylpregna-1, 4-diene-3, 20-dione, is an artificially synthesized glucocorticoid, belongs to a middle-effect glucocorticoid, and has anti-inflammatory, immune mechanism and antiallergic effects. Methylprednisolone can inhibit proliferation of connective tissue, reduce permeability of capillary wall and cell membrane, reduce inflammatory exudation, and inhibit formation and release of histamine and other toxic substances. The retention of water and sodium and the potassium discharging effect of the medicine are smaller than that of prednisone, the medicine has stronger anti-inflammatory and anti-allergic effects and fewer side effects, is mainly used for allergic and inflammatory diseases and organ transplantation to prevent rejection, can also be used as emergency medicine for critical diseases, and has very wide clinical application.
As a glucocorticoid medicine widely applied clinically, methylprednisolone tablets are marketed in the United states in 10/24 of 1957, and are clinically used for rheumatic diseases, collagen diseases, skin diseases, allergic diseases, eye diseases, respiratory diseases, hematopathy, tumors, edema, gastrointestinal diseases, nervous systems, organ transplantation, congenital adrenal hyperplasia, non-suppurative thyroiditis and the like.
The process route for developing the methylprednisolone raw material medicine is as follows:
in the process of manufacturing methylprednisolone, methylprednisolone dehydrogenate generates methylprednisolone iodide through an iodination reaction, and the methylprednisolone iodide is usually methylprednisolone monoiodide (11 beta, 17-dihydroxy-6 alpha-methyl-21-iodopregna-1, 4-diene-3, 20-dione) and methylprednisolone diiodide (11 beta, 17-dihydroxy-6 alpha-methyl-21-iodopregna-1, 4-diene-3, 20-dione) which are mixed in different proportions, have similar polarities and are difficult to separate, so the methylprednisolone iodide is usually directly subjected to the next reaction without being analyzed and separated in the manufacturing process. To date, no literature has reported an analytical method for methylprednisolone iodide intermediates. For the control of methylprednisolone iodide, the complete reaction of the methylprednisolone dehydrogenated substance in the previous step can be ensured, and the side reaction of the methylprednisolone dehydrogenated substance in the subsequent reaction can be controlled, so that the reaction yield is improved, the residue of the methylprednisolone dehydrogenated substance in methylprednisolone acetate and methylprednisolone can be effectively controlled, and therefore, the establishment of a simple and efficient detection and analysis method is necessary for the quality control of the methylprednisolone iodide.
Disclosure of Invention
Aiming at the defect that the prior art lacks a methylprednisolone iodide detection method, the invention provides a methylprednisolone intermediate, and particularly provides a methylprednisolone iodide analysis method. The analysis method provided by the invention has the characteristics of good separation degree, simplicity, rapidness, strong specificity, high sensitivity and the like, and can be used for quality control in the synthetic process of the methylprednisolone raw material medicine and quality evaluation of final products.
The invention provides an analysis method of a methylprednisolone intermediate, which comprises the following steps:
eluting the substance to be detected by high performance liquid chromatography;
the chromatographic conditions are as follows:
the stationary phase is a chromatographic column of octadecylsilane chemically bonded silica;
in the mobile phase, the mobile phase A is water, and the mobile phase B is methanol; the volume ratio of the water to the methanol is 50:50-67: 33;
the substance to be detected comprises methylprednisolone monoiodide and/or methylprednisolone diiodide;
wherein the methylprednisolone monoiodide is 11 beta, 17-dihydroxy-6 alpha-methyl-21-iodopregn-1, 4-diene-3, 20-diketone as shown in formula I,
wherein the methylprednisolone diiodide is 11 beta, 17-dihydroxy-6 alpha-methyl-21, 21-diiodopregna-1, 4-diene-3, 20-diketone as shown in a formula II,
in the analysis method, the volume ratio of the water to the methanol is, for example, 54:46 or 62:38, preferably 57:43 to 67: 33.
In the analysis method, the mobile phase may further comprise an organic acid, and the organic acid is preferably trifluoroacetic acid.
In the analysis method, the ratio of the volume sum of the water and the methanol to the volume of the organic acid is preferably 100:0.008 to 100:0.03, for example 100:0.01, 100: 0.02, preferably 100:0.008 to 100: 0.012.
In the assay, the flow rate of the mobile phase is conventional in the art, and is preferably from 0.5mL/min to 1.5mL/min, more preferably 1.0 mL/min.
In the analysis method, the detection wavelength is preferably 220-250nm, such as 230nm, 240nm, 245nm, 250 nm.
In the analysis method, the conditions used for the chromatographic column are conventional in the art, and the packing particle size of the chromatographic column is preferably 3 to 5 μm, more preferably 5 μm.
In the analysis method, preferably, the chromatographic column is YMC, ODS-A, and the specification is as follows: 15cm 4.6mm, 5 μm.
In the assay, the column temperature of the chromatographic column is conventional in the art, preferably 20-40 ℃, e.g., 20 ℃, 30 ℃, 35 ℃ or 40 ℃.
In the analysis method, the sample injection volume of the chromatographic column is preferably 1-100. mu.L, and more preferably 20. mu.L.
In the analysis method, the substance to be detected can be pretreated before elution so as to meet the sample injection standard. The pretreatment may be a pretreatment conventional in the art, and preferably, the pretreatment comprises the steps of: and dissolving the object to be detected in a solvent. The solvent is preferably methanol.
In the analysis method, preferably, the sample concentration of the analyte is 0.4-0.6mg/mL, such as 0.4mg/mL, 0.5mg/mL or 0.6 mg/mL.
In the analysis method, preferably, the analyte further includes methylprednisolone dehydrogenase, the methylprednisolone dehydrogenase is 6 α -methyl-11 β,17 α -dihydroxypregna-1, 4-diene-3, 20-dione, as shown in formula III,
in the analysis method, preferably, the analyte further comprises methylprednisolone (11 β,17, 21-trihydroxy-6 α -methylpregna-1, 4-diene-3, 20-dione), as shown in formula IV,
in the analysis method, preferably, the substance to be detected is a product obtained by iodination of methylprednisolone dehydrogenated substance. More preferably, the iodine solution is obtained by mixing calcium chloride, iodine and methanol, the methanol, dichloromethane, calcium chloride, methylprednisolone dehydrogenate and calcium oxide are mixed, the iodine solution is added for iodination, after the reaction, an ammonium chloride aqueous solution is added, and the mixture is washed and separated.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the method has the characteristics of good separation degree, simplicity, rapidness, strong specificity, high sensitivity and the like, is suitable for effectively analyzing the methylprednisolone iodide intermediate and impurities thereof, or is further used for monitoring the quality of the methylprednisolone synthesis reaction process, the final product or the raw material medicine and the preparation.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the following examples, the liquid chromatograph used was Shimadzu LC-20 AT.
Preparation example 1: preparation of samples to be tested
In a 25mL single-necked flask, 0.1g of calcium chloride and 10mL of methanol were added, and after dissolution, 1.7g of iodine was added as an iodine solution for future use. A25 mL single-neck flask was charged with 2g of ammonium chloride and 10mL of water to prepare an ammonium chloride solution, which was then used as an ammonium chloride solution. In a 100mL three-necked flask, 20mL of methylene chloride, 0.2g of calcium chloride, 0.3g of calcium oxide, 2g of methylprednisolone dehydrogenation product and 10mL of methanol were added, and an iodine solution was added dropwise to react for about 1 hour. Pumping the prepared ammonium chloride solution into the three-neck flask, and reacting for about 2 hours; and (3) adding 30mL of water into the reaction solution under stirring, filtering, washing with a small amount of water, and collecting a filter cake to obtain methylprednisolone iodide.
Example 1
Chromatographic conditions are as follows:
sample introduction volume: 20 μ l, detection wavelength: 230nm, column: column packed with octadecylsilane chemically bonded silicA (YMC, ODS-A, 15 cm. times.4.6 mm, 5 μm), flow rate: 1ml/min, column temperature: 20 ℃, mobile phase: methanol, water and trifluoroacetic acid in a volume ratio of 62:38:0.01, sample preparation: a proper amount of a detected sample is precisely weighed, dissolved by a proper amount of methanol, diluted to a solution with the concentration of 0.4mg/ml by a mobile phase, shaken up, 20 mu l of the solution is precisely measured and injected into a liquid chromatograph, and the retention time of the chromatogram to a main peak is recorded to be 1.5 times, and the result is shown as follows.
And (2) remarking that the impurity A is methylprednisolone, the impurity B is methylprednisolone dehydrogenation product, the impurity C is unknown impurity with RRT about 0.94, and the impurity D is unknown impurity with RRT about 1.59.
Example 2
Chromatographic conditions are as follows:
sample introduction volume: 20 μ l, detection wavelength: 240nm, column: column packed with octadecylsilane chemically bonded silicA (YMC, ODS-A, 15 cm. times.4.6 mm, 5 μm), flow rate: 1ml/min, column temperature: 30 ℃, mobile phase: methanol, water and trifluoroacetic acid in a volume ratio of 62:38:0.01, sample preparation: a proper amount of a detected sample is precisely weighed, dissolved by a proper amount of methanol, diluted to a solution with the concentration of 0.5mg/ml by a mobile phase, shaken up, 20 mu l of the solution is precisely measured and injected into a liquid chromatograph, and the retention time of the chromatogram to a main peak is recorded to be 2 times, and the result is shown as follows.
Example 3
Chromatographic conditions are as follows:
sample introduction volume: 20 μ l, detection wavelength: 250nm, column: column packed with octadecylsilane chemically bonded silicA (YMC, ODS-A, 15 cm. times.4.6 mm, 5 μm), flow rate: 1ml/min, column temperature: 35 ℃, mobile phase: methanol, water and trifluoroacetic acid in a volume ratio of 62:38:0.01, sample preparation: a proper amount of a detected sample is precisely weighed, dissolved by a proper amount of methanol, diluted to a solution with the concentration of 0.6mg/ml by a mobile phase, shaken up, 20 mu l of the solution is precisely measured and injected into a liquid chromatograph, and the retention time of the chromatogram to a main peak is recorded to be 3 times, and the result is shown as follows.
Example 4
Chromatographic conditions are as follows:
sample introduction volume: 20 μ l, detection wavelength: 245nm, column: column packed with octadecylsilane chemically bonded silicA (YMC, ODS-A, 15 cm. times.4.6 mm, 5 μm), flow rate: 1ml/min, column temperature: 40 ℃, mobile phase: methanol, water and trifluoroacetic acid in a volume ratio of 62:38:0.01, sample preparation: a proper amount of a detected sample is precisely weighed, dissolved by a proper amount of methanol, diluted to a solution with the concentration of 0.5mg/ml by a mobile phase, shaken up, 20 mu l of the solution is precisely measured and injected into a liquid chromatograph, and the retention time of the chromatogram to a main peak is recorded to be 3.5 times, and the result is shown as follows.
Example 5
Chromatographic conditions are as follows:
sample introduction volume: 20 μ l, detection wavelength: 240nm, column: column packed with octadecylsilane chemically bonded silicA (YMC, ODS-A, 15 cm. times.4.6 mm, 5 μm), flow rate: 1ml/min, column temperature: 20 ℃, mobile phase: methanol, water and trifluoroacetic acid in a volume ratio of 54:46:0.01, sample preparation: a proper amount of a detected sample is precisely weighed, dissolved by a proper amount of methanol, diluted to a solution with the concentration of 0.5mg/ml by a mobile phase, shaken up, 20 mu l of the solution is precisely measured and injected into a liquid chromatograph, and the retention time of the chromatogram to a main peak is recorded to be 3 times, and the result is shown as follows.
Example 6
Chromatographic conditions are as follows:
sample introduction volume: 20 μ l, detection wavelength: 230nm, column: column packed with octadecylsilane chemically bonded silicA (YMC, ODS-A, 15 cm. times.4.6 mm, 5 μm), flow rate: 1ml/min, column temperature: 20 ℃, mobile phase: methanol, water and trifluoroacetic acid in a volume ratio of 62:38:0.02, sample preparation: a proper amount of a detected sample is precisely weighed, dissolved by a proper amount of methanol, diluted to a solution with the concentration of 0.4mg/ml by a mobile phase, shaken up, 20 mu l of the solution is precisely measured and injected into a liquid chromatograph, and the retention time of the chromatogram to a main peak is recorded to be 3 times, and the result is shown as follows.
Comparative example 1
Chromatographic conditions are as follows:
sample introduction volume: 20 μ l, detection wavelength: 230nm, column: column packed with octyl silane-bonded silica gel (Agilent, Eclipse Plus C85 μm, 4.6 × 150mm), flow rate: 1ml/min, column temperature: 20 ℃, mobile phase: methanol, water and trifluoroacetic acid in a volume ratio of 62:38:0.01, sample preparation: a proper amount of a detected sample is precisely weighed, dissolved by a proper amount of methanol, diluted to a solution with the concentration of 0.4mg/ml by a mobile phase, shaken up, 20 mu l of the solution is precisely measured and injected into a liquid chromatograph, and the retention time of the chromatogram to a main peak is recorded to be 1.5 times, and the result is shown as follows.
The results show that this method does not achieve an efficient separation of methylprednisolone monoiodide and diiodide.
Comparative example 2
Chromatographic conditions are as follows:
sample introduction volume: 20 μ l, detection wavelength: 230nm, column: column packed with octadecylsilane chemically bonded silicA (YMC, ODS-A, 15 cm. times.4.6 mm, 5 μm), flow rate: 1ml/min, column temperature: 30 ℃, mobile phase: acetonitrile and water in a volume ratio of 60:40, and sample preparation: a proper amount of a detected sample is precisely weighed, dissolved by a proper amount of acetonitrile, diluted to a solution with the concentration of 0.4mg/ml by a mobile phase, shaken up, 20 mu l of the solution is precisely measured and injected into a liquid chromatograph, and the retention time of the chromatogram to a main peak is recorded to be 3 times, and the result is shown as follows.
The results show that this method does not achieve an effective separation of methylprednisolone impurity B, impurity C, monoiodide and diiodide.
Claims (13)
1. The method for analyzing the methylprednisolone intermediate is characterized by comprising the following steps of:
eluting the substance to be detected by high performance liquid chromatography;
the chromatographic conditions are as follows:
the stationary phase is a chromatographic column of octadecylsilane chemically bonded silica;
in the mobile phase, the mobile phase A is water, and the mobile phase B is methanol; the volume ratio of the water to the methanol is 50:50-67: 33;
the substance to be detected comprises methylprednisolone monoiodide and/or methylprednisolone diiodide;
wherein the methylprednisolone monoiodide is 11 beta, 17-dihydroxy-6 alpha-methyl-21-iodopregn-1, 4-diene-3, 20-diketone as shown in formula I,
wherein the methylprednisolone diiodide is 11 beta, 17-dihydroxy-6 alpha-methyl-21, 21-diiodopregna-1, 4-diene-3, 20-diketone as shown in a formula II,
2. the method of analyzing a methylprednisolone intermediate as recited in claim 1, wherein said method of analysis satisfies one or more of the following conditions:
(1) the volume ratio of the water to the methanol is 57:43-67: 33;
(2) the flow rate of the mobile phase is 0.5mL/min-1.5 mL/min;
(3) the detection wavelength is 220-250 nm;
(4) the filler granularity of the chromatographic column is 3-5 mu m;
(5) the temperature of the chromatographic column is 20-40 ℃;
(6) the sample injection volume of the chromatographic column is 1-100 mu L;
(7) the sample injection concentration of the object to be detected is 0.4-0.6 mg/mL;
(8) the substance to be detected also comprises methylprednisolone dehydrogenated substance which is 6 alpha-methyl-11 beta, 17 alpha-dyhydroxyl pregna-1, 4-diene-3, 20-diketone as shown in formula III,
(9) the substance to be detected also comprises methylprednisolone (11 beta, 17, 21-trihydroxy-6 alpha-methylpregna-1, 4-diene-3, 20-diketone) shown in a formula IV,
3. the method of analyzing a methylprednisolone intermediate as recited in claim 1, wherein said method of analysis satisfies one or more of the following conditions:
(1) the volume ratio of the water to the methanol is 54:46 or 62: 38;
(2) the flow rate of the mobile phase is 1.0 mL/min;
(3) the detection wavelength is 230nm, 240nm, 245nm or 250 nm;
(4) the filler particle size of the chromatographic column is 5 mu m;
(5) the column temperature of the chromatographic column is 20 ℃, 30 ℃, 35 ℃ or 40 ℃;
(6) the sample injection volume of the chromatographic column is 20 mu L;
(7) the sample injection concentration of the object to be detected is 0.4mg/mL, 0.5mg/mL or 0.6 mg/mL.
4. The method of analyzing a methylprednisolone intermediate as recited in claim 1, wherein said method of analysis satisfies one or more of the following conditions:
(1) the mobile phase further comprises an organic acid;
(2) the chromatographic column is YMC, ODS-A, and the specification is as follows: 15cm 4.6mm, 5 μm.
5. The method of analyzing methylprednisolone intermediate as claimed in claim 4, wherein said organic acid is trifluoroacetic acid.
6. The method of analyzing methylprednisolone intermediate as claimed in claim 4, wherein the volume ratio of "the sum of the volumes of said water and said methanol" to said organic acid is 100:0.008 to 100: 0.03.
7. The method for analyzing methylprednisolone intermediate as claimed in claim 6, wherein the volume ratio of "the sum of the volumes of said water and said methanol" to said organic acid is 100:0.008 to 100: 0.012.
8. The method of claim 6, wherein the volume ratio of the sum of the volumes of said water and said methanol to said organic acid is 100:0.01 or 100: 0.02.
9. the method for analyzing methylprednisolone intermediate as claimed in claim 1, wherein said analyte is pretreated before elution, and said pretreatment comprises the following steps: and dissolving the object to be detected in a solvent.
10. The method of analyzing methylprednisolone intermediate as claimed in claim 9, wherein said solvent is methanol.
11. The method for analyzing a methylprednisolone intermediate as claimed in claim 1, wherein said analyte is a product obtained by iodination of methylprednisolone dehydrogen.
12. The method for analyzing methylprednisolone intermediate as claimed in claim 11, wherein said analyte is a product obtained by iodination of methylprednisolone dehydrogen.
13. The method of analyzing methylprednisolone intermediate of claim 12, wherein said product is prepared by the following method: mixing calcium chloride, iodine and methanol to obtain iodine solution, mixing methanol, dichloromethane, calcium chloride, methylprednisolone dehydrogenation product and calcium oxide, adding the iodine solution to perform iodination reaction, adding ammonium chloride aqueous solution after the reaction, washing with water, and separating.
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