CN117871730A - Method for simultaneously detecting oxidized impurity and isomer impurity of Fudosteine and application of method - Google Patents
Method for simultaneously detecting oxidized impurity and isomer impurity of Fudosteine and application of method Download PDFInfo
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- CN117871730A CN117871730A CN202410056241.1A CN202410056241A CN117871730A CN 117871730 A CN117871730 A CN 117871730A CN 202410056241 A CN202410056241 A CN 202410056241A CN 117871730 A CN117871730 A CN 117871730A
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- fudosteine
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- 239000012535 impurity Substances 0.000 title claims abstract description 280
- KINWYTAUPKOPCQ-YFKPBYRVSA-N Fudosteine Chemical compound OC(=O)[C@@H](N)CSCCCO KINWYTAUPKOPCQ-YFKPBYRVSA-N 0.000 title claims abstract description 114
- 229950006783 fudosteine Drugs 0.000 title claims abstract description 112
- 238000000034 method Methods 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 claims abstract description 79
- 238000001212 derivatisation Methods 0.000 claims abstract description 46
- 238000002360 preparation method Methods 0.000 claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 claims abstract description 25
- 239000003814 drug Substances 0.000 claims abstract description 25
- 229940079593 drug Drugs 0.000 claims abstract description 22
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 18
- 238000010828 elution Methods 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 239000007787 solid Substances 0.000 claims abstract description 12
- 238000003908 quality control method Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 45
- 239000012488 sample solution Substances 0.000 claims description 38
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims description 26
- 239000000523 sample Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 14
- 239000005695 Ammonium acetate Substances 0.000 claims description 14
- 229940043376 ammonium acetate Drugs 0.000 claims description 14
- 235000019257 ammonium acetate Nutrition 0.000 claims description 14
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 14
- 239000004327 boric acid Substances 0.000 claims description 14
- 238000000926 separation method Methods 0.000 claims description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims description 12
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 claims description 12
- 229940100688 oral solution Drugs 0.000 claims description 11
- 239000007924 injection Substances 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 10
- 239000003826 tablet Substances 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 8
- 239000002775 capsule Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 229960004308 acetylcysteine Drugs 0.000 claims description 6
- 239000008187 granular material Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- 239000012085 test solution Substances 0.000 claims description 5
- 239000012669 liquid formulation Substances 0.000 claims description 4
- 229940054441 o-phthalaldehyde Drugs 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 3
- 239000012088 reference solution Substances 0.000 claims description 3
- 229940088679 drug related substance Drugs 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims 1
- 238000012937 correction Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000003960 organic solvent Substances 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 54
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000011835 investigation Methods 0.000 description 9
- 239000013558 reference substance Substances 0.000 description 9
- 239000002994 raw material Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 230000005526 G1 to G0 transition Effects 0.000 description 6
- XORXDJBDZJBCOC-UHFFFAOYSA-N azanium;acetonitrile;acetate Chemical compound [NH4+].CC#N.CC([O-])=O XORXDJBDZJBCOC-UHFFFAOYSA-N 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 238000010829 isocratic elution Methods 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 150000003457 sulfones Chemical class 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 150000003462 sulfoxides Chemical class 0.000 description 3
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- XXROGKLTLUQVRX-UHFFFAOYSA-N allyl alcohol Chemical compound OCC=C XXROGKLTLUQVRX-UHFFFAOYSA-N 0.000 description 2
- 229960001305 cysteine hydrochloride Drugs 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 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 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N 1-propanol Substances CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 102100028292 Aladin Human genes 0.000 description 1
- 101710065039 Aladin Proteins 0.000 description 1
- 206010003754 Atypical mycobacterial infections Diseases 0.000 description 1
- 206010006448 Bronchiolitis Diseases 0.000 description 1
- 206010006458 Bronchitis chronic Diseases 0.000 description 1
- 208000014085 Chronic respiratory disease Diseases 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000009267 bronchiectasis Diseases 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 208000007451 chronic bronchitis Diseases 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 150000003983 crown ethers Chemical class 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 150000001944 cysteine derivatives Chemical class 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000007919 dispersible tablet Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- 239000007938 effervescent tablet Substances 0.000 description 1
- -1 etc.) Substances 0.000 description 1
- 239000003172 expectorant agent Substances 0.000 description 1
- 230000003419 expectorant effect Effects 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000003885 eye ointment Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000000865 liniment Substances 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 230000000414 obstructive effect Effects 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 206010035653 pneumoconiosis Diseases 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- QWSZRRAAFHGKCH-UHFFFAOYSA-M sodium;hexane-1-sulfonate Chemical compound [Na+].CCCCCCS([O-])(=O)=O QWSZRRAAFHGKCH-UHFFFAOYSA-M 0.000 description 1
- 239000007962 solid dispersion Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000005303 weighing 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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
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Abstract
The invention discloses a method for simultaneously detecting oxidized impurity and Isomer impurity of Fudosteine and application thereof, wherein the detection method creatively uses pre-column derivatization reaction for separating Isomer impurity for separating oxidized impurity, and combines a specific high performance liquid chromatography gradient elution mode and elution condition to realize the technical effects of simultaneously and effectively separating and detecting oxidized impurity YF1, oxidized impurity YF2, oxidized impurity F and Isomer impurity Isomer on a traditional C18 column; in addition, the detection method has high sensitivity, and each impurity correction factor is between 0.2 and 5, is little influenced by auxiliary materials and organic solvents, and is widely applicable to detection of impurities in Fudosteine bulk drugs, solid preparations, semisolid preparations or liquid preparations, thereby improving the overall quality control of the Fudosteine bulk drugs and preparations thereof.
Description
Technical Field
The invention belongs to the technical field of Fudosteine analysis methods, and particularly relates to a method for simultaneously detecting oxidized impurities and isomer impurities of Fudosteine and application thereof.
Background
Fudosteine (2R) -2-amino-3- (3-hydroxypropyl thio) propionic acid is a cysteine derivative, is a novel expectorant, has the functions of inhibiting proliferation of cyclic cells and regulating mucus and mucosa normal state of respiratory tract, and is suitable for treating chronic respiratory diseases (bronchial asthma, chronic bronchitis, bronchiectasis, phthisis, pneumoconiosis, chronic obstructive emphysema, atypical mycobacteriosis, pneumonia, diffuse bronchiolitis and the like). Fudosteine was developed by Mitsubishi pharmaceutical Co., ltd., japan, and dosage forms including tablets and oral solutions were marketed in Japan in 12 months 2001 and 2 months 2004, respectively.
At present, 2 processes for preparing Fudosteine bulk drugs in China are mainly adopted, one process is prepared by reacting cysteine hydrochloride with allyl alcohol, and the other process is prepared by reacting cysteine hydrochloride with 3-halogenated-1-propanol. Fudosteine in domestic market is in form of tablet, capsule, granule or oral liquid. Various impurities exist in fudosteine bulk drugs and formulations, including oxidized impurity YF (sulfoxide impurities including isomers YF1 and YF 2), oxidized impurity F (sulfone impurities) and Isomer impurity Isomer, the impurity generation routes are shown below. The presence of these impurities may have an impact on the safety and effectiveness of the drug product and should be tightly controlled to ensure safe and effective product.
The prior art research on methods for detecting Fudosteine impurities has focused on enantiomeric impurities. The current national drug administration standards (YBH 02592008, YBH02782008, YBH 02752008) for Fudosteine and its formulations in China only control unknown impurities and the dextroisomer of Fudosteine. Literature (Wang Ying, hangtaijun chiral derivatization-reverse phase HPLC method to determine optical purity of Fudosteine [ J ]. Pharmaceutical progress 2005,29 (9): 421-425) isomer impurities were isolated by pre-column derivatization. Patent CN112881538A and patent CN101161642a separate the isomer impurities by chiral stationary phase and chiral mobile phase methods, respectively.
However, the prior art has been silent about methods for detecting oxidized impurities of fudosteine, especially sulfone-based impurities. The literature (Guo Zhiyuan, zhao Xinqing, zhu Hengyi, yuan Jun. HPLC method for measuring the content of related substances in Fudosteine bulk drug and its preparation. Chinese pharmacy, 2019,30 (13): 1764-1769.) discloses a method for detecting sulfoxide impurity C (namely impurity YF) under the condition of ultraviolet wavelength 210nm by taking a traditional C18 column as a chromatographic column and taking a low-pH sodium hexane sulfonate solution as a mobile phase, wherein the method has the defects of weak absorption, low sensitivity, unstable baseline and the like, and can not separate and detect sulfone impurity (namely impurity F).
Further, the research on a method for simultaneously detecting the oxidized impurity and the isomer impurity of fudosteine is more rare. However, the simultaneous detection of oxidized impurities and isomer impurities is critical for the comprehensive evaluation of the quality of fudosteine drug substance and its formulation. The patent CN112881538A adopts a chiral stationary phase method to simultaneously separate and detect the impurity A (namely the impurity YF), the impurity B (namely the impurity F) and the dextroisomer (namely the impurity Isomer) so as to solve the problems that oxidized impurities remain weaker and cannot be effectively separated in a C18 column or an amino column in the prior art. However, the chiral stationary phase using silica gel coated with chiral crown ether as a filler is easily damaged by organic solvents other than methanol and potassium ions, so that the application range of the chiral stationary phase is limited, and the chiral stationary phase is not particularly suitable for detecting oral solutions containing the organic solvents and various soluble auxiliary materials.
Therefore, under the specific condition that the traditional C18 column is adopted as the chromatographic column, a proper method is selected, so that oxidized impurities and isomer impurities of Fudosteine can be separated and detected at the same time, and the technical problem which is not solved by the person skilled in the art is solved.
Disclosure of Invention
The invention aims to solve the technical problems that: under the specific condition that a traditional C18 column is adopted as a chromatographic column, the oxidation impurity YF1, the oxidation impurity YF2, the oxidation impurity F and the Isomer impurity Isomer of Fudosteine are simultaneously and effectively separated and detected by combining a pre-column derivatization reaction with specific high performance liquid chromatography conditions (elution mode, elution condition and the like), so that the quality control of the Fudosteine bulk drug and the preparation thereof is improved.
Aiming at the technical problems to be solved, the invention is solved by the following technical scheme:
a method for simultaneously detecting oxidized impurity and Isomer impurity of Fudosteine adopts a pre-column derivatization reaction combined with high performance liquid chromatography, and simultaneously separates and detects oxidized impurity YF, oxidized impurity F and Isomer impurity Isomer.
Preferably, the method for simultaneously detecting the oxidized impurity and the isomer impurity of the fudosteine provided by the invention comprises the following steps of:
step one: sample processing
Diluting Fudosteine with water to prepare a sample solution and a reference solution;
step two: pre-column derivatization reactions
Dissolving phthalic dicarboxaldehyde and N-acetylcysteine in boric acid solution to prepare a derivatization reagent; measuring the sample solutions obtained in the first step, uniformly mixing, and completely derivatizing;
step three: high performance liquid chromatography detection
Taking each sample solution after derivatization, and sampling the sample solution into a chromatograph for detection, wherein the setting of detection conditions comprises:
chromatographic column: octadecylsilane chemically bonded silica column;
mobile phase: consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.03-0.07 mol/L ammonium acetate, and the mobile phase B is acetonitrile; gradient elution, the ratio of mobile phases a and B is as follows:
alternatively, the ratio of mobile phases a and B is as follows:
detection wavelength: 340nm;
the column temperature is 20-30 ℃;
the flow rate is 1.3-1.7 mL/min.
Preferably, in the first step, the concentration of Fudosteine in the sample solution is 8 mug/mL-4 mg/mL.
Preferably, in the second step, the dosage ratio of the phthalic dicarboxaldehyde to the N-acetylcysteine is 1:1.
Preferably, in the second step, the pH of the boric acid solution is 10.2.+ -. 0.1.
More preferably, in step two, the derivatizing reagent is 6mmol/L of phthalic aldehyde and 6mmol/L N-acetylcysteine in 0.04mol/L boric acid solution.
More preferably, the volume ratio of the derivatizing reagent prepared in the second step to the sample solution obtained in the first step is 5:1.
More preferably, the derivatization reagent prepared in the second step is uniformly mixed with the sample solution obtained in the first step to carry out derivatization reaction, and the mixture is kept stand for 10 to 20 minutes until the reaction is complete.
Preferably, in the third step, the setting of the detection condition further includes: the sample injection volume is 25-50 mu L.
Preferably, in the third step, the setting of the detection condition further includes: the chromatographic column is WatersHSS T3 (4.6X105 mm,5 μm) or model Agilent ZORBAX SB-Aq (4.6X105 mm,5 μm), or other equivalent performance columns.
Preferably, in the third step, the setting of the detection condition includes: the mobile phase A was 0.05mol/L ammonium acetate, the column temperature was 25℃and the flow rate was 1.5mL/min.
In the aforementioned method for simultaneously detecting oxidized impurity and Isomer impurity of fudosteine, the structures of oxidized impurity YF, oxidized impurity F and Isomer impurity Isomer are respectively:
the invention further provides application of the method for simultaneously detecting the oxidized impurity and the isomer impurity of the fudosteine, which is used for detecting the impurity in the fudosteine bulk drug and the fudosteine preparation (including but not limited to solid preparation, semisolid preparation and liquid preparation).
The invention further provides application of the method for simultaneously detecting the oxidized impurity and the isomer impurity of the Fudosteine, which is used for quality control in research and development or production of Fudosteine raw material medicines and Fudosteine preparations (including but not limited to solid preparations, semisolid preparations and liquid preparations) so as to ensure that the impurity is not overrun.
Preferably, the aforementioned detection method can be used for detecting impurities in a fudosteine liquid preparation (especially an oral solution), and comprises the following steps:
step one: sample processing
Dissolving Fudosteine oral solution in water, and quantitatively diluting to obtain solution containing 4mg/mL of Fudosteine in 1mL, wherein the solution is used as a test solution; precisely measuring a proper amount of sample solution, adding water to dilute the sample solution to prepare a solution with the concentration of 0.2 percent equivalent to the sample solution, and taking the solution as a control solution;
step two: pre-column derivatization reactions
6mmol/L o-phthalaldehyde and 6mmol/L N-acetylcysteine are dissolved in 0.04mol/L boric acid solution to prepare a derivatization reagent; precisely measuring 0.2mL of each sample solution in the first step, respectively adding 1mL of derivatization reagent, uniformly mixing, and standing for 20min;
step three: high performance liquid chromatography detection
Precisely measuring each sample solution after the derivatization reaction in the second step, and injecting the sample solution into a chromatograph for detection, wherein the setting of detection conditions comprises:
chromatographic column: agilent ZORBAX SB-Aq, 4.6X105 mm,5 μm or a column with comparable performance;
mobile phase: ammonium acetate of 0.05mol/L is taken as a mobile phase A, pure acetonitrile is taken as a fluidity B, and elution is carried out according to the following gradient:
detection wavelength: 340nm;
column temperature: 25 ℃;
flow rate: 1.5mL/min;
sample injection volume: 25. Mu.L.
The Fudosteine solid preparation comprises, but is not limited to, granules, capsules, tablets, powder, films, solid dispersions and the like; fudosteine semisolid formulations referred to herein include, but are not limited to ointments, creams, eye ointments, gels, and the like; fudosteine liquid formulations referred to herein include, but are not limited to, oral solutions, drops, suspensions, injections, liniments, lotions and the like.
That is, the invention provides 2 methods for simultaneously detecting oxidized impurity and Isomer impurity of specific Fudosteine, which can be widely used in Fudosteine bulk drug, various solid preparations, semisolid preparations or liquid preparations of Fudosteine, and can detect oxidized impurity YF (including Isomer YF1 and YF 2), oxidized impurity F and Isomer impurity Isomer in the preparations, and control the quality of Fudosteine bulk drug or the preparations thereof so as to improve the stability of the bulk drug or the preparations, ensure that the impurity is not overrun and accord with the drug evaluation.
Compared with the prior art, the method for simultaneously detecting the oxidized impurity and the isomer impurity of Fudosteine has the beneficial effects that:
(1) the invention creatively uses the pre-column derivatization reaction for separating the Isomer impurities for separating the oxidized impurities, and combines a specific high performance liquid chromatography gradient elution mode and elution conditions, thereby realizing the technical effects of simultaneously and effectively separating and detecting the oxidized impurities YF (comprising isomers YF1 and YF 2), the oxidized impurities F and the Isomer impurities Isomer on the traditional C18 column.
(2) The method has high detection sensitivity, the intensity correction factor of each impurity absorption peak is between 0.2 and 5, the content of each impurity can be calculated by adopting a main component self-dilution contrast method, and the defects of low impurity absorption peak and correction factor of >7 of the undeivatized reaction and the need of calculating the impurity content by adopting an external standard method in the prior art are overcome.
(3) The invention can separate oxidized impurity and isomer impurity simultaneously and effectively on the basis of the traditional C18 column, has smaller influence by auxiliary materials and organic solvents and wider application range compared with the chiral stationary phase method in the prior art, can be used for the comprehensive quality control of Fudosteine bulk drugs, solid preparations, semisolid preparations and liquid preparations (especially granules, capsules, tablets, oral solutions and the like), and is especially suitable for Fudosteine oral solutions.
Drawings
Fig. 1 is a graph of the detection results of the method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine provided in example 1.
Fig. 2 is a graph of the detection results of the method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine provided in example 2.
FIG. 3 is a graph showing the results of detection of the mobile phase (1) condition in the mobile phase type test of example 3.
FIG. 4 is a graph showing the results of detection of the mobile phase (2) condition in the mobile phase type test of example 3.
FIG. 5 is a graph showing the results of a single impurity YF control under mobile phase (2) condition in mobile phase type investigation in example 3.
FIG. 6 is a graph showing the results of the single impurity F control under mobile phase (2) condition in mobile phase type investigation in example 3.
FIG. 7 is a graph showing the results of the mobile phase (3) condition detection in the mobile phase ratio test of example 4.
FIG. 8 is a graph showing the results of the single impurity YF control under mobile phase (3) condition in mobile phase ratio investigation of example 4.
FIG. 9 is a graph showing the results of the single impurity F control under mobile phase (3) condition in mobile phase ratio investigation of example 4.
Fig. 10 is a test result of the test method of example 6 for detecting impurities in oral solutions.
In fig. 1 to 10, the definition of each peak mark is as follows:
1. f is an oxidized impurity F of sulfones;
2. YF-1 and YF-2 refer to sulfoxide oxidized impurities YF, which have 2 isomers and are correspondingly marked as YF-1 and YF-2;
3. YGT refers to an Isomer impurity Isomer;
4. FDST refers to the raw material Fudosteine;
5. YSSJ refers to derivatizing agents.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and examples. The scope of the invention includes but is not limited to these embodiments, and all changes and equivalents that do not depart from the spirit of the invention are intended to be included therein.
The type and manufacturer information of the used instruments and experimental reagents involved in all the embodiments of the invention are as follows:
instrument:
waters Arc high performance liquid chromatograph
Reagent:
fudosteine oral solution (50 mL:4g, toosendan pharmaceutical Co., ltd.);
fudosteine control (purity 99.5%, middle school);
oxidized impurity YF (purity 99.24%, ap);
oxidizing impurity F (97.70%, ap);
isomer impurity Isomer (96.41%, QCC);
phthalic aldehyde (AR, aladin);
n-acetylcysteine (AR, sigma);
boric acid (AR, national drug);
sodium hydroxide (AR, chinese medicine);
ammonium acetate (AR, national drug);
acetonitrile (HPLC, TEDIA).
Example 1A method for simultaneous detection of oxidized and isomeric impurities of Fudosteine
The embodiment provides a method for simultaneously detecting oxidized impurity and isomer impurity of Fudosteine, which comprises the following steps:
step one: sample processing
Accurately weighing proper amounts of Fudosteine, the impurity Isomer, the impurity F and the impurity YF reference substances, adding water for dissolution and quantitatively diluting to prepare a system applicability solution containing 4mg of Fudosteine, 80 mug of the impurity Isomer, 8 mug of the impurity F and 8 mug of the impurity YF in each 1 mL.
Step two: pre-column derivatization reactions
6mmol/L o-phthalaldehyde and 6mmol/L N-acetylcysteine are dissolved in 0.04mol/L boric acid solution (4.96 g boric acid and 2.28g sodium hydroxide are taken, water is added to dissolve and dilute the mixture to 200mL, and 2mol/L NaOH solution is used for regulating the pH value to 10.2+/-0.1, so that the derivatization reagent (derivative reagent) is prepared;
precisely measuring 0.2mL of each sample solution in the first step, respectively adding 1mL of derivatization reagent, uniformly mixing, and standing for 10-20 min.
Step three: high performance liquid chromatography detection
Precisely measuring each sample solution after the derivatization reaction in the second step, and injecting the sample solution into a high performance liquid chromatograph for detection, wherein the detection conditions are as follows:
chromatographic column: watersHSS T3 (4.6X105 mm,5 μm) or other column of comparable potency;
mobile phase: ammonium acetate of 0.05mol/L is taken as a mobile phase A, pure acetonitrile is taken as a fluidity B, and elution is carried out according to the following gradient:
detection wavelength: 340nm;
column temperature: 25 ℃;
flow rate: 1.5mL/min;
sample injection volume: 50. Mu.L.
The detection results are shown in FIG. 1 and Table 1, and the impurity YF has 2 isomer impurities YF1 and YF2; the impurities F, YF, YF2 are both oxidized impurities, and the impurity Isomer is an Isomer impurity.
TABLE 1 detection results of Fudosteine impurity of EXAMPLE 1
| Peak name | Retention time (min) | Peak area | Peak height | Degree of separation |
| Impurity F | 24.457 | 138691 | 2200 | / |
| Impurity YF1 | 26.257 | 85391 | 1331 | 1.80 |
| Impurity YF2 | 31.347 | 133605 | 1715 | 4.57 |
| Fudosteine | 42.483 | 82736780 | 714812 | 7.29 |
| Impurity Isomer | 45.763 | 1567410 | 35075 | 2.57 |
From the test results of fig. 1 and table 1, it is shown that:
the peak retention times of the oxidized impurities F, the oxidized impurities YF1, the oxidized impurities YF2, the Fudosteine and the Isomer impurities Isomer are 24.457min, 26.257min, 31.347min, 42.483min and 45.763min respectively, and the separation degrees are all more than 1.5, so that the effective separation of the oxidized impurities and the Isomer impurities can be realized simultaneously.
In addition, only the concentration of Fudosteine and each impurity in the system applicability solution of the first step was replaced to be 8 mug/mL, other steps are as described above, and the detected results also show that: the main peak and each impurity peak are strong in absorption, and the detection method is high in sensitivity; and the correction factors of the impurities are all between 0.2 and 5, and the content of the impurities can be calculated by adopting a main component self-dilution contrast method.
Example 2 another method for simultaneous detection of oxidized and isomeric impurities of Fudosteine
The present embodiment provides another method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine, comprising the following steps:
step one: as in example 1.
Step two: as in example 1.
Step three: high performance liquid chromatography detection
Precisely measuring each sample solution after the derivatization reaction in the second step, and injecting the sample solution into a high performance liquid chromatograph for detection, wherein the detection conditions are as follows:
chromatographic column: agilent ZORBAX SB-Aq (4.6X105 mm,5 μm) or a column of comparable performance;
mobile phase: ammonium acetate of 0.05mol/L is taken as a mobile phase A, pure acetonitrile is taken as a fluidity B, and elution is carried out according to the following gradient:
detection wavelength: 340nm;
column temperature: 25 ℃;
flow rate: 1.5ml/min;
sample injection volume: 25. Mu.L.
The detection results are shown in fig. 2 and table 2, and it is known that impurity YF has 2 isomer impurities YF1 and YF2; the impurities F, YF, YF2 are both oxidized impurities, and the impurity Isomer is an Isomer impurity.
TABLE 2 detection results of Fudosteine impurity of EXAMPLE 2
| Peak name | Retention time (min) | Peak area | Degree of separation |
| Impurity F | 8.080 | 71279 | / |
| Impurity YF1 | 9.310 | 59299 | 2.22 |
| Impurity YF2 | 10.458 | 65716 | 1.89 |
| Fudosteine | 22.655 | 39730262 | 15.35 |
| Impurity Isomer | 24.443 | 524943 | 2.97 |
| Derivatization reagent peak | 25.721 | 131020 | 1.68 |
From the test results of fig. 2 and table 2, it is shown that:
the separation degree of the oxidized impurities F, the oxidized impurities YF1, the oxidized impurities YF2, the Fudosteine and the Isomer impurities Isomer is more than 1.5, and the derivatization reagent peaks are not overlapped with other Fudosteine impurities (oxidized impurities F, oxidized impurities YF1, oxidized impurities YF2 and Isomer impurities Isomer), so that the effective separation of the oxidized impurities and the Isomer impurities can be realized simultaneously.
In addition, only the concentration of Fudosteine and each impurity in the system applicability solution of the first step was replaced to be 8 mug/mL, other steps are as described above, and the detected results also show that: the main peak and each impurity peak are strong in absorption, and the detection method is high in sensitivity; and the correction factors of the impurities are all between 0.2 and 5, and the content of the impurities can be calculated by adopting a main component self-dilution contrast method.
Example 3 influence factor investigation of mobile phase species
Under the condition of isocratic elution, the influence of different mobile phase types on separation of oxidized impurities and isomer impurities is examined, and the examination process is as follows:
step one: pre-column derivatization reaction:
one or more of Fudosteine, isomer impurity Isomer reference substance, oxidized impurity YF reference substance and oxidized impurity YF reference substance are dissolved in water to respectively prepare different sample solutions. The derivatization reagent and the derivatization reaction conditions were the same as in example 2.
Step two: high performance liquid chromatography detection
The detection conditions were set as follows:
chromatographic column: agilent ZORBAX SB-Aq, 4.6X105 mm,5 μm;
mobile phase: (1) 23mmol/L acetate buffer (pH 6.0) -methanol-acetonitrile (60:37:3), or (2) 0.05mol/L ammonium acetate-acetonitrile (90:10);
flow rate: 1.5mL/min;
detection wavelength: 340nm;
column temperature: 25 ℃;
sample injection volume: 25. Mu.L.
The detection results are shown in fig. 3, 4, 5, 6 and table 3, and it is known that the impurity YF has 2 isomer impurities YF1 and YF2; the impurities F, YF, YF2 are both oxidized impurities, and the impurity Isomer is an Isomer impurity.
TABLE 3 results of investigation of mobile phase species under isocratic elution
When 23mmol/L acetate buffer (pH 6.0) -methanol-acetonitrile (60:37:3) was used as mobile phase (1)), the results from the assays of FIG. 3 and Table 3 show that:
1) Oxidized impurities YF1, YF2 and F all appear at a peak in 2.775 min;
2) Fudosteine and Isomer impurity Isomer both peak at 4.028 min;
that is, neither oxidized nor isomer impurities can be separated effectively.
When 0.05mol/L ammonium acetate-acetonitrile (90:10) (i.e., mobile phase (2)) was used as the mobile phase, the results from the measurements of FIGS. 4, 5, 6 and Table 3 showed that:
1) Fudosteine has a resolution of >1.5 from the Isomer impurity Isomer, see FIG. 4;
2) Peaks of oxidized impurities YF1, YF2 and F overlap, and the peak condition appears at 2.077min in FIG. 4; in addition, FIG. 5 is a result spectrum of a single impurity YF reference under the condition of the mobile phase (2), FIG. 6 is a result spectrum of a single impurity F reference under the condition of the mobile phase (2), and the phenomenon that peaks of the oxidized impurities overlap is further demonstrated by the peak-out conditions of the impurities YF-1, YF-2 and F in FIG. 5 and FIG. 6;
that is, oxidized impurities cannot be separated efficiently, but isomer impurities can be separated efficiently.
It follows that the chromatographic conditions of the prior art cannot be referenced to separate both the oxidized and the isomeric impurities, and even the optimized mobile phase species can only separate the isomeric impurities, and the oxidized impurities cannot be separated.
Example 4 influence factor investigation of mobile phase ratio
Under the condition of isocratic elution, the influence of different flow phase ratios on separation of oxidized impurities and isomer impurities is examined, and the examination process is as follows:
step one: pre-column derivatization reactions
One or more of Fudosteine, isomer impurity Isomer reference substance, oxidized impurity YF reference substance and oxidized impurity YF reference substance are dissolved in water to respectively prepare different sample solutions. The derivatization reagent and the derivatization reaction conditions were the same as in example 2.
Step two: high performance liquid chromatography detection
The detection conditions were set as follows:
chromatographic column: agilent ZORBAX SB-Aq, 4.6X105 mm,5 μm;
mobile phase: (2) 0.05mol/L ammonium acetate-acetonitrile (90:10), or (3) 0.05mol/L ammonium acetate-acetonitrile (95:5);
flow rate: 1.5mL/min;
detection wavelength: 340nm;
column temperature: 25 ℃;
sample injection volume: 25. Mu.L.
The detection results are shown in fig. 4, 5, 6, 7, 8, 9 and table 4, and it is understood that the impurity YF has 2 isomer impurities YF1 and YF2; the impurities F, YF, YF2 are both oxidized impurities, and the impurity Isomer is an Isomer impurity.
TABLE 4 results of mobile phase proportion investigation under isocratic elution
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When 0.05mol/L ammonium acetate-acetonitrile (90:10) (i.e., mobile phase (2)) was used as the mobile phase, the oxidized impurities could not be separated effectively, but the isomer impurities could be separated effectively, and the specific analysis was examined under the condition of mobile phase (2) of example 3.
When the mobile phase was 0.05mol/L ammonium acetate-acetonitrile (95:5) (i.e., mobile phase (3)), the detection results from FIGS. 7, 8, 9 and Table 4 showed that:
1) Fudosteine has a degree of separation from the Isomer impurity Isomer of >1.5, but the Isomer impurity Isomer overlaps the derivatization reagent peak, see FIG. 7;
2) Oxidized impurity F and YF1 peaks overlap, see table 4; in addition, fig. 8 is a result spectrum of a single impurity YF reference under the condition of the mobile phase (3), fig. 9 is a result spectrum of a single impurity F reference under the condition of the mobile phase (3), and the phenomenon that the peaks of the oxidized impurities overlap is further confirmed by the peak-out conditions of the impurities YF-1 and F in fig. 8 and 9.
It follows that optimizing the mobile phase ratio under isocratic elution conditions still fails to separate oxidized impurities and the risk of overlapping derivatizing reagent peaks increases as the organic phase ratio decreases.
In summary, the analysis of examples 3 and 4 in combination with the detection results of examples 1 and 2 revealed that the simultaneous and effective separation detection of oxidized impurities and isomer impurities could be achieved only under the specific gradient elution conditions of examples 1 or 2.
Example 5 durability inspection of example 1 and example 2
On the basis of determining the gradient elution conditions described in example 1 and example 2, the high performance liquid chromatography column temperature, the flow rate and the ammonium acetate concentration were examined for durability, the column temperature range was examined to be 20-30 ℃, the flow rate range was examined to be 1.3-1.7 mL/min, and the ammonium acetate concentration was examined to be 0.03-0.07 mol/L.
The examination results are shown in Table 5, and the impurity YF has 2 isomer impurities YF1 and YF2; the impurities F, YF, YF2 are both oxidized impurities, and the impurity Isomer is an Isomer impurity.
TABLE 5 durability investigation results of Fudosteine impurity detection of example 2
From the test results in table 5, it is shown that:
under the conditions that the column temperature is 20-30 ℃, the flow rate is 1.3-1.7 mL/min and the ammonium acetate concentration is 0.03-0.07 mol/L, the separation degree of each impurity is more than 1.5, and the derivatization reagent peaks are not overlapped with other Fudosteine impurities (oxidized impurity F, oxidized impurity YF1, oxidized impurity YF2 and Isomer impurity Isomer), which shows that the detection methods of the embodiment 1 and the embodiment 2 are good in durability.
Example 6 application example of Fudosteine bulk drug and impurity detection in its preparation
As described above, the method for simultaneously detecting the oxidized impurity and the isomer impurity of the Fudosteine can be used for detecting the impurity in the Fudosteine bulk drug and the preparation thereof and controlling the quality.
In this example, an oral solution of Fudosteine is taken as an example, and the implementation process of the detection method provided by the invention is described. The specific detection steps are as follows:
step one: sample processing
Dissolving Fudosteine oral solution in water, and quantitatively diluting to obtain solution containing 4mg/mL of Fudosteine in 1mL, wherein the solution is used as a test solution; precisely measuring a proper amount of the sample solution, adding water to dilute the sample solution to prepare a solution with the concentration of 0.2 percent equivalent to the sample solution, and taking the solution as a reference substance solution.
Step two: pre-column derivatization reactions
Dissolving 6mmol/L phthalic aldehyde and 6mmol/L N-acetylcysteine in 0.04mol/L boric acid solution (4.96 g boric acid and 2.28g sodium hydroxide are taken, water is added to dissolve and dilute the solution to 200mL, and 2mol/L NaOH solution is used for regulating the pH value to 10.2+/-0.1, so that the derivatization reagent is prepared;
precisely measuring 0.2mL of the sample solution and the reference substance solution in the first step, respectively adding 1mL of the derivatization reagent, uniformly mixing, and standing for 10-20 min.
Step three: high performance liquid chromatography detection
Precisely measuring each sample solution after the derivatization reaction in the second step, and injecting the sample solution into a high performance liquid chromatograph for detection, wherein the detection conditions are as follows:
chromatographic column: agilent ZORBAX SB-Aq (4.6X105 mm,5 μm) or a column of comparable performance;
mobile phase: ammonium acetate of 0.05mol/L is taken as a mobile phase A, pure acetonitrile is taken as a fluidity B, and elution is carried out according to the following gradient:
detection wavelength: 340nm;
column temperature: 25 ℃;
flow rate: 1.5ml/min;
sample injection volume: 25. Mu.L.
The detection results are shown in FIG. 10 and Table 5, and the impurity YF has 2 isomer impurities YF1 and YF2; the impurities F, YF, YF2 are both oxidized impurities, and the impurity Isomer is an Isomer impurity.
TABLE 5 impurity detection results for Fudosteine oral solution of EXAMPLE 6
| Peak name | Retention time (min) | Degree of separation | Impurity content (%) |
| Impurity F | 9.299 | / | Not detected |
| Impurity YF1 | 10.196 | / | 0.08 |
| Impurity YF2 | 11.440 | 2.02 | 0.13 |
| Fudosteine | 21.970 | 16.50 | 99.43 |
| Impurity Isomer | 23.007 | 2.62 | 0.36 |
From the detection results of fig. 10 and table 5, it is shown that: the separation degree of the main peak and each impurity peak is more than 1.5, so that each impurity is effectively separated; the content of each impurity is calculated according to the self-dilution contrast method of the main component and is respectively as follows: the impurity F was not detected, the impurity YF1 content was 0.08%, the impurity YF2 content was 0.13%, and the impurity Isomer content was 0.36%.
It should be noted that, in this embodiment, only the oral solution of Fudosteine is taken as an example, and the application of the detection method provided by the invention is described by way of example, and the method of embodiment 2 is particularly suitable for detecting impurities in a sample which is stable for a long time. However, the practical application range is not meant to be limited to oral solutions, and the detection methods described in examples 1 and 2 can be extended to be applied to the raw material drug of Fudosteine, and other various solid, semisolid or liquid formulations of Fudosteine, so long as the preparation contains oxidized impurity YF (including isomers YF1 and YF 2), oxidized impurity F and Isomer impurity Isomer, and quality control of the raw material drug of Fudosteine or the preparation thereof is performed to improve stability of the raw material or the preparation, ensure that the impurities do not exceed limits, and meet the drug evaluation rules.
In particular, the method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine described in example 1 and example 2 can be applied to detection of impurity in fudosteine bulk drug, tablet (including but not limited to common tablet, dispersible tablet, effervescent tablet, etc.), granule and capsule. Wherein,
when the method is used for detecting the impurity of the raw material medicine, the first step can be as follows:
and (3) dissolving the fudosteine raw material medicine in water, quantitatively diluting to prepare a solution containing 4mg/mL of fudosteine in each 1mL of solution, taking the solution as a test solution, and detecting impurities in the fudosteine oral solution in other steps.
When the method is used for detecting impurity substances of Fudosteine granules, capsules and tablets, the first step can be as follows:
grinding and crushing Fudosteine particles, capsule contents or tablets, adding water for dissolving and quantitatively diluting to prepare a solution containing 4mg/mL of Fudosteine in each 1mL, filtering to obtain a supernatant serving as a test solution, and detecting impurities in the Fudosteine oral solution in other steps.
Claims (10)
1. A method for simultaneously detecting oxidized impurity and Isomer impurity of fudosteine is characterized in that a pre-column derivatization reaction is combined with high performance liquid chromatography, and oxidized impurity YF, oxidized impurity F and Isomer impurity Isomer are simultaneously detected in a separation mode; the derivatization reagent used in the pre-column derivatization reaction is a mixed solution obtained by dissolving phthalic dicarboxaldehyde and N-acetylcysteine in a boric acid solution; the high performance liquid chromatography condition uses gradient elution, and the mobile phase consists of ammonium acetate and acetonitrile.
2. The method for simultaneous detection of oxidized impurity and isomer impurity of fudosteine according to claim 1, comprising the steps of:
step one: sample processing
Diluting Fudosteine with water to prepare a sample solution and a reference solution;
step two: pre-column derivatization reactions
Dissolving phthalic dicarboxaldehyde and N-acetylcysteine in boric acid solution to prepare a derivatization reagent; measuring the sample solutions obtained in the first step, uniformly mixing, and completely derivatizing;
step three: high performance liquid chromatography detection
Taking each sample solution after derivatization, and sampling the sample solution into a chromatograph for detection, wherein the setting of detection conditions comprises:
chromatographic column: octadecylsilane chemically bonded silica column;
mobile phase: consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.03-0.07 mol/L ammonium acetate, and the mobile phase B is acetonitrile; gradient elution, the ratio of mobile phases a and B is as follows:
alternatively, the ratio of mobile phases a and B is as follows:
detection wavelength: 340nm;
the column temperature is 20-30 ℃;
the flow rate is 1.3-1.7 mL/min.
3. The method for simultaneous detection of oxidized and isomeric impurities according to claim 2, wherein in said step one, the concentration of fudosteine in the sample solution is from 8 μg/mL to 4mg/mL.
4. The method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine according to claim 2, wherein the ratio of the amount of phthalic dicarboxaldehyde to the amount of N-acetylcysteine in the second step is 1:1, or the pH of the boric acid solution in the second step is 10.2±0.1.
5. The method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine according to claim 4, wherein in the second step, the derivatization reagent is 6mmol/L o-phthalaldehyde and 6mmol/L N-acetylcysteine dissolved in 0.04mol/L boric acid solution; preferably, the volume ratio of the derivatization reagent prepared in the second step to the sample solution obtained in the first step is 5:1; more preferably, the mixture is left to stand for 10 to 20 minutes until the derivatization reaction is completed.
6. The method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine according to claim 2, wherein in said step three, the setting of the detection conditions further comprises: the sample injection volume is 25-50 mu L.
7. The method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine according to claim 2, wherein in said step three, the setting of the detection conditions further comprises: the chromatographic column is 4.6X1250 mm long, has a particle size of 5 μm and is a Waters typeHSS T3, or a column having a length of 4.6X105 mm, a particle size of 5 μm, and a model number of Agilent ZORBAX SB-Aq, or a column having a comparable performance.
8. The method for simultaneously detecting oxidized impurity and isomer impurity of fudosteine according to claim 2, wherein in said step three, the setting of the detection conditions comprises: the mobile phase A was 0.05mol/L ammonium acetate, the column temperature was 25℃and the flow rate was 1.5mL/min.
9. Use of the method for simultaneous detection of oxidized and isomeric impurities of fudosteine according to any one of claims 1 to 8, for the detection of impurities in fudosteine bulk drugs, solid preparations, semi-solid preparations and liquid preparations; or for quality control in the development or production of fudosteine drug substances, solid, semi-solid and liquid formulations; preferably, the solid preparation of Fudosteine comprises, but is not limited to, granules, capsules and tablets; fudosteine liquid formulations include, but are not limited to, oral solutions.
10. Use of the method for simultaneous detection of oxidized and isomeric impurities of fudosteine according to claim 9, characterized in that it comprises the following steps when used for detecting impurities in an oral solution of fudosteine:
step one: sample processing
Dissolving Fudosteine oral solution in water, and quantitatively diluting to obtain solution containing 4mg/mL of Fudosteine in 1mL, wherein the solution is used as a test solution; precisely measuring a proper amount of sample solution, adding water to dilute the sample solution to prepare a solution with the concentration of 0.2% equivalent to the sample solution, and taking the solution as a reference solution;
step two: pre-column derivatization reactions
6mmol/L o-phthalaldehyde and 6mmol/L N-acetylcysteine are dissolved in 0.04mol/L boric acid solution to prepare a derivatization reagent; precisely measuring 0.2mL of each sample solution in the first step, respectively adding 1mL of derivatization reagent, uniformly mixing, and standing for 20min;
step three: high performance liquid chromatography detection
Precisely measuring each sample solution after the derivatization reaction in the second step, and injecting the sample solution into a chromatograph for detection, wherein the setting of detection conditions comprises:
chromatographic column: agilent ZORBAX SB-Aq, 4.6X105 mm,5 μm or a column with comparable performance;
mobile phase: ammonium acetate of 0.05mol/L is taken as a mobile phase A, pure acetonitrile is taken as a fluidity B, and elution is carried out according to the following gradient:
detection wavelength: 340nm;
column temperature: 25 ℃;
flow rate: 1.5mL/min;
sample injection volume: 25. Mu.L.
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