CN105486785A - Method for detecting enantiomer impurity in apremilast - Google Patents
Method for detecting enantiomer impurity in apremilast Download PDFInfo
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- CN105486785A CN105486785A CN201510982153.5A CN201510982153A CN105486785A CN 105486785 A CN105486785 A CN 105486785A CN 201510982153 A CN201510982153 A CN 201510982153A CN 105486785 A CN105486785 A CN 105486785A
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- Prior art keywords
- apremilast
- butyl ether
- methyl tert
- enantiomter
- mobile phase
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- IMOZEMNVLZVGJZ-QGZVFWFLSA-N apremilast Chemical compound C1=C(OC)C(OCC)=CC([C@@H](CS(C)(=O)=O)N2C(C3=C(NC(C)=O)C=CC=C3C2=O)=O)=C1 IMOZEMNVLZVGJZ-QGZVFWFLSA-N 0.000 title claims abstract description 115
- 229960001164 apremilast Drugs 0.000 title claims abstract description 114
- 238000000034 method Methods 0.000 title claims abstract description 44
- 239000012535 impurity Substances 0.000 title description 7
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 238000012360 testing method Methods 0.000 claims description 75
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 68
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 54
- 239000013558 reference substance Substances 0.000 claims description 37
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 27
- 102000012404 Orosomucoid Human genes 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 12
- 150000001298 alcohols Chemical class 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000012046 mixed solvent Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 2
- 108010061952 Orosomucoid Proteins 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- 238000011084 recovery Methods 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 229920002472 Starch Polymers 0.000 abstract description 4
- 239000008107 starch Substances 0.000 abstract description 4
- 235000019698 starch Nutrition 0.000 abstract description 4
- 239000011248 coating agent Substances 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 67
- 239000000945 filler Substances 0.000 description 37
- -1 methyl tert-butyl Chemical group 0.000 description 31
- 230000014759 maintenance of location Effects 0.000 description 29
- 239000000523 sample Substances 0.000 description 27
- 238000000926 separation method Methods 0.000 description 26
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 19
- 239000000741 silica gel Substances 0.000 description 17
- 229910002027 silica gel Inorganic materials 0.000 description 17
- 239000002245 particle Substances 0.000 description 16
- 238000004305 normal phase HPLC Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 11
- 239000007924 injection Substances 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 238000004811 liquid chromatography Methods 0.000 description 8
- HCTNDDRMUNVGSU-UHFFFAOYSA-N ethanol;2-methoxy-2-methylpropane Chemical compound CCO.COC(C)(C)C HCTNDDRMUNVGSU-UHFFFAOYSA-N 0.000 description 7
- GCFHZZWXZLABBL-UHFFFAOYSA-N ethanol;hexane Chemical compound CCO.CCCCCC GCFHZZWXZLABBL-UHFFFAOYSA-N 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- ZXKXJHAOUFHNAS-FVGYRXGTSA-N (S)-fenfluramine hydrochloride Chemical compound [Cl-].CC[NH2+][C@@H](C)CC1=CC=CC(C(F)(F)F)=C1 ZXKXJHAOUFHNAS-FVGYRXGTSA-N 0.000 description 5
- 238000010812 external standard method Methods 0.000 description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 235000010980 cellulose Nutrition 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 2
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 2
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 2
- 239000008108 microcrystalline cellulose Substances 0.000 description 2
- 229940016286 microcrystalline cellulose Drugs 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WSVLPVUVIUVCRA-KPKNDVKVSA-N Alpha-lactose monohydrate Chemical compound O.O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O WSVLPVUVIUVCRA-KPKNDVKVSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 150000004696 coordination complex Chemical class 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- UMYZHWLYICNGRQ-UHFFFAOYSA-N ethanol;heptane Chemical compound CCO.CCCCCCC UMYZHWLYICNGRQ-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229940035429 isobutyl alcohol Drugs 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229960001375 lactose Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229960001021 lactose monohydrate Drugs 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 229940011530 otezla Drugs 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—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
- G01N30/60—Construction of the column
Landscapes
- 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)
- Spectroscopy & Molecular Physics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a method for detecting an enantiomer (I) in apremilast. A protein type chiral chromatographic column is adopted in the method; while the defect of shorter service life of an existing starch coating chiral column is overcome, the enantiomer in the apremilast is effectively separated and detected, and a detection result is accurate and reliable; the method has the multiple advantages such as excellent linear relation, high precision, good stability, good repeatability, good recovery rate, simple and convenient operation and low cost, and is suitable for popularization and application. A structural formula is shown in the description.
Description
Technical field
The present invention relates to the detection method of enantiomter impurity in Apremilast.
Background technology
Apremilast (apremilast, (S)-2-[1-(3-ethoxy-4-methoxyphenyl)-2-methylsulfonylethyl]-4-acetylaminoisoindoline-1,3-diketone, molecular formula: C
22h
24n
2o
7s, molecular weight: be 460.5) developed by Celgene biotech company of the U.S., approval in Nikkei U.S. food Drug Administration March 21 in 2014 (FDA) is for the treatment of active type psoriasis arthropathica (PsA) that be grown up, and commodity are called Otezla.
Apremilast is a kind of chiral drug, and it is effectively configured as S configuration.Therefore, in Apremilast product, the existence of its R configurational isomer impurity (I) can have a strong impact on drug safety and the clinical effectiveness of product, is necessary to detect impurity (I) content in Apremilast, with the quality of effective monitoring Apremilast.
Chinese patent CN104458961A (publication date 2015.03.25) discloses a kind of method of Apremilast related substance, the method uses AD-H chromatographic column, mobile phase is normal hexane/isopropyl alcohol/diethylamine/trifluoroacetic acid=80/20/0.2/0.5, flow velocity 1.0mL/min, column temperature 40 DEG C, determined wavelength 240nm.In patent CN104792913A (publication date 2015.07.22), its method is verified, find that the degree of separation of Apremilast and its enantiomter body only has 1.26, be less than 1.5, completely both are not separated.Visible, the method cannot the content of enantiomter in Accurate Determining Apremilast.
Chinese patent CN104792913A (publication date 2015.07.22), CN105136933A (publication date 2015.12.09), CN104820028A (publication date 2015.08.05) and one section of document (cover invention, HPLC method measures Apremilast enantiomter, chemical intermediate, 03 phase in 2015,37-38 page) all disclose the method for separating and detecting of Apremilast enantiomter, wherein CN104792913A and CN105136933A adopts is AD-H chiral chromatographic column, and CN104820028A and aforementioned documents adopt is AS-H chiral chromatographic column.
Visible, in the method for existing report, the chiral column of employing all belongs to cellulose type chiral chromatographic column, and is all made up of starch in replace cellulose.But, because starch is water miscible, must the absolute guarantee pillar life-span in mobile phase.Inevitably, after using the long period, such chromatographic column can suffer damage, and serviceable life is shorter.
In addition, the shortcoming of such chromatographic column to be used for positive, and expensive, in use procedure, careless slightly will cause the post of chromatographic column imitate reduce, blocking even can not use.
Therefore, be necessary to search out low price, usable range is wider, and accurately can detect the other types chiral chromatographic column of enantiomter impurity in Apremilast.
Summary of the invention
For solving the problem, the invention provides a kind of detection method utilizing enantiomter (I) in the Apremilast of protein type chiral chromatographic column, it comprises the following steps:
A, prepare need testing solution:
Get Apremilast sample to be measured, mobile phase dissolves, and filters or does not filter, preparing need testing solution;
The reference substance solution of b, preparation enantiomter (I):
Get enantiomter (I) reference substance, mobile phase dissolves, and prepares the reference substance solution of enantiomter (I);
C, respectively need testing solution and reference substance solution are injected high performance liquid chromatograph and detect, chromatographic condition is as follows:
Namely chromatographic column: acid seromucoid chiral chromatographic column is the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
Mobile phase: the mixed solvent of methyl tert-butyl ether and C1 ~ C4 alcohols solvent; Described C1 ~ C4 alcohols solvent comprises methyl alcohol, ethanol, propyl alcohol, isopropyl alcohol, normal butyl alcohol, isobutyl alcohol and the tert-butyl alcohol.
Determined wavelength: 230 ± 10nm, preferred determined wavelength is 230 ± 2nm;
D, calculate the content of enantiomter (I) in Apremilast sample according to testing result.In specific embodiment of the present invention, employing be external standard method calculate.
Described Apremilast sample can be selected from the tablet or other formulation that Apremilast raw material or Apremilast obtain.
Further, in step c, the specification of described chromatographic column is: internal diameter 4.6mm, length 150mm, packing material size 5 μm.Further, in step c, the model of described chromatographic column is CHIRALAGP.
Further, in step c, described C1 ~ C4 alcohols solvent is ethanol, isopropyl alcohol or normal butyl alcohol.
Further, in step c, the volume ratio of described methyl tert-butyl ether and alcohols solvent is 70%:30% ~ 90%:10%.
Further, in step c, described mobile phase is the mixed solvent of following volume ratio:
Methyl tert-butyl ether: normal butyl alcohol=70:30, methyl tert-butyl ether: normal butyl alcohol=75:25, methyl tert-butyl ether: normal butyl alcohol=90:10, methyl tert-butyl ether: ethanol=70:30, methyl tert-butyl ether: ethanol=75:25, methyl tert-butyl ether: ethanol=90:10, normal heptane: ethanol=90:10, methyl tert-butyl ether: isopropyl alcohol=70:30, methyl tert-butyl ether: isopropyl alcohol=75:25 or methyl tert-butyl ether: isopropyl alcohol=90:10; Preferable methyl tertbutyl ether: normal butyl alcohol=75:25.
Further, in step a, the concentration of need testing solution is 0.1 ~ 0.3mg/mL; In step b, the concentration of reference substance solution is 0.1 ~ 1.0 μ g/mL.
Further, in step c, the column temperature of described chromatographic condition is 30 DEG C ~ 40 DEG C, and preferred column temperature is 30 DEG C ~ 35 DEG C; Flow velocity is 0.8mL/min ~ 1.2mL/min, and preferred flow velocity is 0.8mL/min ~ 1.0mL/min.
Further, in step c, described mobile phase is methyl tert-butyl ether: ethanol, isopropyl alcohol or normal butyl alcohol volume ratio are the mixed solvent of 75:25, and the column temperature of described chromatographic condition is 30 DEG C, and flow velocity is 0.8mL/min.
Further, in step c, the sample size of described chromatographic condition is 20 μ L.
The detection method of enantiomter in Apremilast of the present invention, employing be protein type chiral chromatographic column, usable range is comparatively wide, and price is lower, and serviceable life is longer.While overcoming the life-span shorter point of existing starch coated type chiral column, effectively stage enantiomer separation in Apremilast is detected, testing result accurately, reliably, and there is the plurality of advantages such as linear relationship excellence, precision are high, good stability, reproducible, the recovery good, easy and simple to handle, cost is low, be suitable for applying.
Meanwhile, the inventive method adopt the chiral chromatographic column of protein type.
And, the taxonomic hierarchies according to the chiral chromatographic column that IrvingWainer proposes based on the interaction mechanism of chiral stationary phase and solvent:
1st class: form compound by hydrogen bond, π-π effect, even level-even level effect.
, there is again embedding compound in the 2nd class: the interaction in existing Class1.
3rd class: enter chirality hole based on solvent and form embedding compound.
4th class: based on the metal complex forming diastereomer.
5th class: realize chiral separation based on hydrophobic interaction and polar interaction.
Cellulose type chiral chromatographic column in existing report belongs to typical 2nd class chiral column, and protein type chiral chromatographic column provided by the invention belongs to the 5th class.Visible, protein type chiral chromatographic column provided by the invention is also the enantiomter impurity accurately detected in Apremilast, provides a brand-new chromatographic column and selects approach.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 is that embodiment 1 the inventive method is to the testing result of system suitability.
Fig. 2 is the testing result of comparative example's 1 pair of mixed solution.
Fig. 3 is the canonical plotting of embodiment 11.
Fig. 4-6 is the testing result of embodiment 11, and Fig. 4 is (a) need testing solution; Fig. 5 is (b) reference substance solution; Fig. 6 is (c) negative sample solution.
Embodiment
The raw material used in the specific embodiment of the invention, equipment are known product, obtain by buying commercially available prod.
High performance liquid chromatograph (model: LC-20AT binary pump, manufacturer: Japanese Shimadzu Corporation);
Electronic balance (model: AUW220D, manufacturer: Japanese Shimadzu Corporation).
Apremilast sheet (lot number: 140501, source: the abundant Pharmacy stock Co., Ltd in Chengdu hundred)
Enantiomter reference substance (lot number: PCL-A012, source: Mai Sike Pharmaceutical Technology Co., Ltd)
Embodiment 1
1, the determination of determined wavelength
Get Apremilast sheet, porphyrize, precision takes in right amount, is mixed with the solution that concentration is 20 μ g/mL, as need testing solution with mobile phase (methyl tert-butyl ether-normal butyl alcohol=75:25).
It is appropriate that precision takes reference substance (enantiomter), is mixed with the solution that concentration is 20 μ g/mL, product solution in contrast with mobile phase (methyl tert-butyl ether-normal butyl alcohol=75:25).
Get above-mentioned need testing solution, reference substance solution, scan in 200nm ~ 400nm wavelength coverage, test findings is in table 1.
Table 1, UV scanning result of the present invention
| Project | Spike long (nm) | Peak value | Paddy wavelength (nm) | Valley |
| Need testing solution | 230 | 0.617 | 215 | 0.226 |
| Reference substance solution | 230 | 0.775 | 296 | 0.010 |
Test findings shows, determined wavelength, within the scope of 220nm ~ 240nm, is all applicable to high performance liquid chromatography of the present invention and detects.
2, system suitability:
Get Apremilast, each 20 μ g of enantiomter, add 1mL mobile phase (normal hexane-ethanol=75:25), as system suitability solution; Measure 20 μ Lindenmayer system applicability solution injection liquid chromatographies, record chromatogram, the degree of separation between Apremilast and enantiomter should be not less than 2.0, and theoretical cam curve calculates should be not less than 2000 by Apremilast peak.
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-normal butyl alcohol (volume ratio is 75:25);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
The testing result of system suitability is shown in Fig. 1, degree of separation between Apremilast (retention time is 6.353min) and enantiomter (retention time is 5.345min) is 3.654, and theoretical cam curve (calculating by Apremilast peak) is 7349.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
3, high performance liquid chromatography detects
Prepare need testing solution: take Apremilast sheet, porphyrize, take 100mg, be placed in 50mL measuring bottle, add mobile phase (methyl tert-butyl ether-normal butyl alcohol=75:25) appropriate, jolting 20 minutes, adds mobile phase and is diluted to scale, shake up, filter, get subsequent filtrate as need testing solution.
Preparation reference substance solution: take enantiomter (I) reference substance 2mg, be placed in 100mL measuring bottle, precision measures 1mL, be placed in 100mL measuring bottle, add mobile phase (methyl tert-butyl ether-normal butyl alcohol=75:25) and be diluted to scale, shake up, in contrast product solution.
Respectively need testing solution and reference substance solution are injected high performance liquid chromatograph to detect, adopt Normal-phase HPLC to detect, chromatographic condition is as follows:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-normal butyl alcohol (volume ratio is 75:25);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
According to testing result, the content calculating enantiomter in Apremilast sheet according to external standard method is 0.03%.
Comparative example embodiment 1
Reversed-phase high-performance liquid chromatography is adopted to detect:
Related substance detection method according to Apremilast sheet carries out from plan quality standard:
The filling agent of chromatographic column: be filling agent (InertsilODS3,50 × 4.6mm, 5 μm or the suitable chromatographic column of other performances) with octadecylsilane chemically bonded silica;
Mobile phase: with 0.05mol/L potassium dihydrogen phosphate-methyl alcohol (50:50) for mobile phase;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Mixed solution: get Apremilast, each 20 μ g of enantiomter, add 1mL mobile phase, as mixed solution;
Prepare need testing solution: get Apremilast sheet, porphyrize, take 100mg, be placed in 50mL measuring bottle, add mobile phase appropriate, jolting 20 minutes, adds mobile phase and is diluted to scale, shake up, and filters, gets subsequent filtrate as need testing solution.
Preparation reference substance solution: take reference substance 2mg, be placed in 100mL measuring bottle, precision measures 1mL, is placed in 100mL measuring bottle, adds mobile phase and is diluted to scale, shake up, in contrast product solution.
The testing result of mixed solution as shown in Figure 2.
The chromatographic peak of result display Apremilast and enantiomter overlaps, and this chromatographic condition accurately can not detect the enantiomter in Apremilast.
Comparative example embodiment 2
Normal-phase HPLC is adopted to detect:
The filling agent of chromatographic column: be the chromatographic column of filling agent with the silica gel of amylose-three (chloro-4 methyl phenyl carbamates of 3-) bonding;
The model of chromatographic column is CHIRALPAKIF, and specification is: internal diameter is 4.6mm, and length is 250mm, and filling agent particle diameter is 5 μm;
Mobile phase: normal hexane-ethanol (75:25);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 7.017min) and enantiomter (supplying its retention time to be 6.579min) is 1.485, theoretical cam curve (calculating by Apremilast peak) is 4721, the tailing factor at Apremilast peak is 1.281, main peak symmetry is poor, the baseline fluctuation of this chromatographic system is larger, degree of separation between main peak and isomeride is less than 1.5, does not meet the requirement of States Pharmacopoeia specifications.
This chromatographic condition accurately can not detect the enantiomter in Apremilast.
Comparative example 3
Normal-phase HPLC is adopted to detect:
The filling agent of chromatographic column: be the chromatographic column of filling agent with the silica gel of amylose-three (chloro-4 methyl phenyl carbamates of 3-) bonding;
The model of chromatographic column is CHIRALPAKIF, and specification is: internal diameter is 4.6mm, and length is 250mm, and filling agent particle diameter is 5 μm;
Mobile phase: normal hexane-ethanol (90:10);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 4.012min) and enantiomter (supplying its retention time to be 3.871min) is 0.721, theoretical cam curve (calculating by Apremilast peak) is 7713, the tailing factor at Apremilast peak is 1.052, baseline fluctuation is large, degree of separation between main peak and isomeride is less than 1.5, does not meet the requirement of States Pharmacopoeia specifications.
This chromatographic condition accurately can not detect the enantiomter in Apremilast.
Embodiment 2
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-normal butyl alcohol (75:25);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 6.353min) and enantiomter (supplying its retention time to be 5.345min) is 3.654, theoretical cam curve (calculating by Apremilast peak) is 7349, and the tailing factor at Apremilast peak is 0.975.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
Embodiment 3
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-normal butyl alcohol (70:30);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 10.115min) and enantiomter (supplying its retention time to be 8.725min) is 4.128, theoretical cam curve (calculating by Apremilast peak) is 8107, and the tailing factor at Apremilast peak is 1.08.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
Embodiment 4
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-normal butyl alcohol (90:10);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 5.011min) and enantiomter (supplying its retention time to be 4.323min) is 1.951, theoretical cam curve (calculating by Apremilast peak) is 12037, and the tailing factor at Apremilast peak is 1.007.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
Embodiment 5
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-ethanol (70:30);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 9.228min) and enantiomter (supplying its retention time to be 8.011min) is 3.055, theoretical cam curve (calculating by Apremilast peak) is 8877, and the tailing factor at Apremilast peak is 1.035.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
Embodiment 6
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-ethanol (90:10);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 5.517min) and enantiomter (supplying its retention time to be 4.229min) is 1.876, theoretical cam curve (calculating by Apremilast peak) is 11039, and the tailing factor at Apremilast peak is 0.982.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
Embodiment 7
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-isopropyl alcohol (70:30);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 17.813min) and enantiomter (supplying its retention time to be 16.055min) is 3.379, theoretical cam curve (calculating by Apremilast peak) is 9527, and the tailing factor at Apremilast peak is 1.028.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
Embodiment 8
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-isopropyl alcohol (90:10);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 10.803min) and enantiomter (supplying its retention time to be 8.058min) is 4.121, theoretical cam curve (calculating by Apremilast peak) is 8859, and the tailing factor at Apremilast peak is 1.056.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
Embodiment 9
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-isopropyl alcohol (75:25);
Column temperature: 30 DEG C;
Flow velocity: 0.8mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 8.757min) and enantiomter (supplying its retention time to be 6.023min) is 8.147, theoretical cam curve (calculating by Apremilast peak) is 7133, and the tailing factor at Apremilast peak is 1.039.
Visible, this chromatographic condition accurately can detect the enantiomter in Apremilast.
Embodiment 10
Normal-phase HPLC is adopted to detect:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-isopropyl alcohol (75:25);
Column temperature: 40 DEG C;
Flow velocity: 1.2mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
System suitability is carried out according to the method for embodiment 1, result shows: the degree of separation between Apremilast (retention time is 5.503min) and enantiomter (supplying its retention time to be 4.727min) is 2.513, theoretical cam curve (calculating by Apremilast peak) is 9586, the tailing factor at Apremilast peak is 0.998, and Normal-phase HPLC of the present invention may be used for detecting the enantiomter in Apremilast.
Embodiment 11 chromatographic condition screening of the present invention
1, mobile phase shaker test
Select different mobile phases to carry out shaker test, other chromatographic conditions are as follows:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: table 2;
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
Carry out system suitability according to the method for embodiment 1, testing result is in table 2:
Table 2, mobile phase shaker test result
| Numbering | Mobile phase | Retention time | Degree of separation | Tailing factor | Theoretical cam curve |
| 1 | Methyl tertiary butyl ether-normal butyl alcohol (75:25) | 6.353min | 3.654 | 0.975 | 7349 |
| 2 | Methyl tertiary butyl ether-normal butyl alcohol (70:30) | 10.115min | 4.128 | 1.08 | 8107 |
| 3 | Methyl tertiary butyl ether-normal butyl alcohol (90:10) | 5.011min | 1.951 | 1.007 | 12037 |
| 4 | Methyl tertiary butyl ether-ethanol (70:30) | 9.228min | 3.055 | 1.035 | 8877 |
| 5 | Methyl tertiary butyl ether-ethanol (90:100) | 5.517min | 1.876 | 0.982 | 11039 |
| 6 | Methyl tertiary butyl ether-isopropyl alcohol (70:30) | 17.813min | 3.379 | 1.028 | 9527 |
| 7 | Methyl tertiary butyl ether-isopropyl alcohol (90:10) | 10.803min | 4.121 | 1.056 | 8859 |
| 8 | Normal hexane-ethanol (70:30) | 9.581min | 1.812 | 1.387 | 3278 |
| 9 | Normal hexane-ethanol (75:25) | 7.017min | 1.485 | 1.281 | 4721 |
| 10 | Normal hexane-ethanol (90:10) | 4.012min | 0.721 | 1.052 | 7713 |
In table 2, mobile phase " such as: methyl tert-butyl ether-normal butyl alcohol (75:25) " has the implication that Chinese Pharmacopoeia version in 2015 has usually, and wherein 75:25 represents the volume ratio of methyl tert-butyl ether and normal butyl alcohol; Retention time refers to the retention time of Apremilast; Degree of separation refers to the degree of separation between Apremilast and enantiomter; Tailing factor refers to the tailing factor at Apremilast peak; Theoretical cam curve refers to theoretical cam curve (calculating by Apremilast peak).
Test findings shows, mobile phase is methyl tert-butyl ether-normal butyl alcohol (70:30), methyl tert-butyl ether-normal butyl alcohol (75:25), methyl tert-butyl ether-normal butyl alcohol (90:10), methyl tert-butyl ether-ethanol (70:30), methyl tert-butyl ether-ethanol (75:25), methyl tert-butyl ether-ethanol (90:10) normal heptane-ethanol (90:10), methyl tert-butyl ether-isopropyl alcohol (70:30), methyl tert-butyl ether-isopropyl alcohol (75:25), time methyl tert-butyl ether-isopropyl alcohol (90:10), all be suitable for detecting the enantiomter in Apremilast.
2, column temperature and flow velocity shaker test
Select different column temperatures and flow velocity to carry out shaker test, other chromatographic conditions are as follows:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-normal butyl alcohol (75:25);
Column temperature: table 3;
Flow velocity: table 3;
Determined wavelength: 230nm;
Sample size: 20 μ L.
Carry out system suitability according to the method for embodiment 1, testing result is in table 3:
Table 3, column temperature and flow velocity shaker test result
In table 3, retention time refers to the retention time of Apremilast; Degree of separation refers to the degree of separation between Apremilast and enantiomter; Tailing factor refers to the tailing factor at Apremilast peak; Theoretical cam curve refers to theoretical cam curve (calculating by Apremilast peak).
Test findings shows, under different column temperatures and flow velocity, the inventive method all can effectively accurately detect Apremilast isomeride.Preferred column temperature is 30 DEG C ~ 35 DEG C; Flow velocity is 0.8mL/min ~ 1.0mL/min.
The Method validation of embodiment 12 the inventive method
In Method validation, chromatographic condition is all as follows:
Chromatographic column: be the chromatographic column of filling agent with the silica gel of acid seromucoid bonding;
The model of chromatographic column is CHIRALAGP, and specification is: internal diameter is 4.6mm, and length is 150mm, and filling agent particle diameter is 5 μm;
Mobile phase: methyl tert-butyl ether-normal butyl alcohol (75:25);
Column temperature: 35 DEG C;
Flow velocity: 1.0mL/min;
Determined wavelength: 230nm;
Sample size: 20 μ L.
1, linear relationship
Prepare reference substance solution according to the inventive method, make the reference substance solution of a series of concentration in table 4 with mobile phase dilution.According to above-mentioned chromatographic condition, detect respectively, testing result is in table 4.
The testing result of table 4, linear relationship test
| Numbering | 1 | 2 | 3 | 4 | 5 |
| Concentration (μ g/mL) | 0..0993 | 0.1987 | 0.3973 | 0.4967 | 0.7947 |
| Peak area | 11383 | 22220 | 42160 | 51929 | 82506 |
With isomer concentration X for horizontal ordinate, peak area Y is ordinate, carries out linear regression, obtains regression equation and is: Y=10179X+1593, R
2=0.999.
Test findings shows, the concentration of isomeride is within the scope of 0.0993 μ g/mL ~ 0.7947 μ g/mL, and its peak area is good linear relationship with measuring concentration.
2, specificity
Need testing solution, reference substance solution is prepared according to the inventive method.
Water intaking lactose 10g, cross-linked carboxymethyl fiber sodium 1g, microcrystalline cellulose 2.1g, dolomol 0.5g, above four kinds of auxiliary materials, mixing; Negative sample solution is prepared according to similar approach.
Sample size: 20 μ L.
Precision measures above-mentioned each solution 20 μ L injection liquid chromatography, and record chromatogram, is shown in Fig. 4-6, is respectively: (a) need testing solution; (b) reference substance solution; (c) negative sample solution.
Test findings shows, negative sample solution can not cause interference to the method for enantiomter in Normal-phase HPLC detection Apremilast of the present invention.
3, precision
Get reference substance enantiomter appropriate, accurately weighed, add mobile phase and dissolve and quantitatively dilute the solution made containing 0.2 μ g in every 1mL, product solution in contrast;
Precision measures 20 μ L reference substance solution, injection liquid chromatography, continuous sample introduction 6 times, record chromatogram.
Testing result is in table 5.
The testing result of table 5, precision test
Test findings shows, the inventive method precision is good.
4, stability
Get Apremilast sheet, be mixed with need testing solution and mapping reference substance solution according to the inventive method, get need testing solution respectively at 0h, 2h, 4h, 6h, 8h sample detection; Precision measures 20 μ L reference substance solution, and injection liquid chromatography, by external standard method with the content of enantiomter in calculated by peak area test sample, the results are shown in Table 6.
The testing result of table 6, stability test
Test findings shows, the need testing solution of the inventive method preparation has good stability.
5, repeatability
Get Apremilast sheet, be mixed with need testing solution according to the inventive method, respectively parallel preparation 6 parts of need testing solutions, and prepare mapping reference substance solution; Precision measures 20 μ L reference substance solution and need testing solutions, and injection liquid chromatography, by external standard method with the content of enantiomter in calculated by peak area test sample, the results are shown in Table 7 respectively.
The testing result of table 7, replica test
Test findings shows, the inventive method reproducible.
6, chromatographic condition and system suitability
Get Apremilast and enantiomter appropriate, add mobile phase and make solution respectively containing 20 μ g in every 1mL, as system suitability solution; Measure 20 μ Lindenmayer system applicability solution injection liquid chromatographies, record chromatogram, the degree of separation between Apremilast and enantiomter should be not less than 2.0, and theoretical cam curve calculates should be not less than 2000 by Apremilast peak.
Prepare need testing solution: get Apremilast sheet, porphyrize, take 100mg, be placed in 50mL measuring bottle, add mobile phase (methyl tert-butyl ether-normal butyl alcohol=75:25) appropriate, jolting 20 minutes, adds mobile phase and is diluted to scale, shake up, filter, get subsequent filtrate as need testing solution.
Preparation reference substance solution: take reference substance (enantiomter) 2mg, be placed in 100mL measuring bottle, precision measures 1mL, be placed in 100mL measuring bottle, add mobile phase (methyl tert-butyl ether-normal butyl alcohol=75:25) and be diluted to scale, shake up, in contrast product solution.
Get reference substance solution 20 μ L, injection liquid chromatography, regulate detection sensitivity, the peak height of enantiomter chromatographic peak is made to be about 20% of registering instrument full scale, precision measures need testing solution and each 20 μ L of reference substance solution again, respectively injection liquid chromatography, record chromatogram.If show enantiomter peak in the chromatogram of need testing solution, must not 0.1% be crossed by external standard method with the content of calculated by peak area enantiomter.According to above-mentioned method, respectively detection is carried out 3 times to the Apremilast sheet of two kinds of different sizes (specification: 10mg, 30mg).
Test findings shows, in test sample, the testing result of enantiomter is not all more than 0.1%, and Apremilast and enantiomter reach good baseline separation.
7, quantitative limit and detectability
Take enantiomter 5.07mg, be placed in 25mL measuring bottle, add mobile phase and dissolve and be settled to scale, shake up, in contrast product solution, precision measures 1.0mL, be placed in 100mL measuring bottle, add mobile phase and dissolve and be settled to scale, shake up, precision measures 1mL again, puts in 20mL measuring bottle, adds mobile phase and dissolves and be settled to scale, shake up, then precision measures 1mL, put in 20mL measuring bottle, add mobile phase dissolve and be settled to scale, shake up, as the detection solution (S/N ≈ 10) of quantitative limit test; Precision measures 3mL, puts in 10mL measuring bottle, adds mobile phase and dissolves and be settled to scale, shake up, as the detection solution (S/N ≈ 3) of detectability test;
Record enantiomter and be quantitatively limited to 4.965ng/mL.
Record enantiomter detection and be limited to 1.490ng/mL.
8, the recovery
Concentration 1: get lactose monohydrate 10g, cross-linked carboxymethyl fiber sodium 1g, microcrystalline cellulose 2.1g, the above-mentioned four kinds of auxiliary materials and mixing of dolomol 0.5g, take about 0.1g, be placed in 50mL measuring bottle, add Apremilast enantiomter reference substance stock solution (concentration: 4.0046 μ g/mL) 2mL, solubilizer is diluted to scale, shakes up, filter, get subsequent filtrate, to obtain final product, prepare three parts with method; Concentration 2: get the blank auxiliary prepared in prescription ratio, take about 0.1g, be placed in 50mL measuring bottle, add Apremilast enantiomter reference substance stock solution (concentration: 4.0046 μ g/mL) 2.5mL, solubilizer is diluted to scale, shakes up, filter, get subsequent filtrate, to obtain final product, prepare three parts with method; Concentration 3: get the blank auxiliary prepared in prescription ratio, take about 0.1g, be placed in 50mL measuring bottle, add Apremilast enantiomter reference substance stock solution (concentration: 4.0046 μ g/mL) 3mL, solubilizer is diluted to scale, shakes up, filter, get subsequent filtrate, to obtain final product, prepare three parts with method.In addition, prepare isomer control product solution according to the above-mentioned method drafted, accurate absorption need testing solution and isomer control product solution 20 μ L sample introduction measure, record chromatogram, and calculate measured amount and the recovery of mentioned component, and it the results are shown in Table 8.
Table 8 enantiomter recovery test result table
Above test findings shows, this method measures the content of isomeride, and the recovery is better, and the relative standard deviation of the recovery of mensuration is less, and accuracy is higher.
In sum, the detection method of enantiomter in Apremilast of the present invention, stage enantiomer separation in Apremilast is detected, testing result accurately, reliably, and there is the plurality of advantages such as linear relationship excellence, precision are high, good stability, reproducible, the recovery good, easy and simple to handle, cost is low, be suitable for applying.
Claims (11)
1. the detection method of enantiomter (I) in Apremilast, is characterized in that: it comprises the following steps:
A, prepare need testing solution:
Get Apremilast sample to be measured, mobile phase dissolves, and filters or does not filter, preparing need testing solution;
The reference substance solution of b, preparation enantiomter (I):
Get enantiomter (I) reference substance, mobile phase dissolves, and prepares the reference substance solution of enantiomter (I);
C, respectively need testing solution and reference substance solution are injected high performance liquid chromatograph and detect, chromatographic condition is as follows:
Chromatographic column: acid seromucoid chiral chromatographic column;
Mobile phase: the mixed solvent of methyl tert-butyl ether and C1 ~ C4 alcohols solvent;
Determined wavelength: 230 ± 10nm, preferred determined wavelength is 230 ± 2nm;
D, calculate the content of enantiomter (I) in Apremilast sample according to testing result.
2. detection method according to claim 1, is characterized in that: in step c, and the specification of described chromatographic column is: internal diameter 4.6mm, length 150mm, packing material size 5 μm.
3. detection method according to claim 2, is characterized in that: in step c, and the model of described chromatographic column is CHIRALAGP.
4. the detection method according to any one of claim 1-3, is characterized in that: in step c, and described C1 ~ C4 alcohols solvent is ethanol, isopropyl alcohol or normal butyl alcohol.
5. detection method according to claim 4, is characterized in that: in step c, and the volume ratio of described methyl tert-butyl ether and alcohols solvent is 70%:30% ~ 90%:10%.
6. detection method according to claim 5, is characterized in that: in step c, and described mobile phase is the mixed solvent of following volume ratio:
Methyl tert-butyl ether: normal butyl alcohol=70:30, methyl tert-butyl ether: normal butyl alcohol=75:25, methyl tert-butyl ether: normal butyl alcohol=90:10, methyl tert-butyl ether: ethanol=70:30, methyl tert-butyl ether: ethanol=75:25, methyl tert-butyl ether: ethanol=90:10, normal heptane: ethanol=90:10, methyl tert-butyl ether: isopropyl alcohol=70:30, methyl tert-butyl ether: isopropyl alcohol=75:25 or methyl tert-butyl ether: isopropyl alcohol=90:10; Preferable methyl tertbutyl ether: normal butyl alcohol=75:25.
7. detection method according to claim 1, is characterized in that: in step a, and the concentration of need testing solution is 0.1 ~ 0.3mg/mL; In step b, the concentration of reference substance solution is 0.1 ~ 1.0 μ g/mL.
8. the method according to any one of claim 1-7, is characterized in that: in step c, and the column temperature of described chromatographic condition is 30 DEG C ~ 40 DEG C, and preferred column temperature is 30 DEG C ~ 35 DEG C; Flow velocity is 0.8mL/min ~ 1.2mL/min, and preferred flow velocity is 0.8mL/min ~ 1.0mL/min.
9. the detection method according to any one of claim 4-8, it is characterized in that: in step c, described mobile phase is methyl tert-butyl ether: ethanol, isopropyl alcohol or normal butyl alcohol volume ratio are the mixed solvent of 75:25, and the column temperature of described chromatographic condition is 30 DEG C, and flow velocity is 0.8mL/min.
10. the detection method according to any one of claim 1-9, is characterized in that: in step c, and the sample size of described chromatographic condition is 20 μ L.
11. detection methods according to any one of claim 1-10, is characterized in that: described Apremilast sample is Apremilast tablet.
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| CN112305107A (en) * | 2020-10-23 | 2021-02-02 | 杭州朱养心药业有限公司 | Apremilast composition of phosphodiesterase-4 inhibitor and quality detection method |
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