CN116148399B - High performance liquid chromatography for separating and detecting oxcarbazepine starting material and related substances thereof - Google Patents

High performance liquid chromatography for separating and detecting oxcarbazepine starting material and related substances thereof Download PDF

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CN116148399B
CN116148399B CN202310421319.0A CN202310421319A CN116148399B CN 116148399 B CN116148399 B CN 116148399B CN 202310421319 A CN202310421319 A CN 202310421319A CN 116148399 B CN116148399 B CN 116148399B
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solution
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acetonitrile
monoammonium phosphate
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CN116148399A (en
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尚文燕
赵娜
王宏
牛玉乐
张燕
李艳平
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Jilin Huikang Pharmaceutical Co ltd
Beijing Sihuan Pharmaceutical Co Ltd
Beijing Ao He Research Institute Co Ltd
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Jilin Huikang Pharmaceutical Co ltd
Beijing Sihuan Pharmaceutical Co Ltd
Beijing Ao He Research Institute Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—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
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention provides a high performance liquid chromatography for separating and detecting oxcarbazepine starting materials and related substances thereof, wherein a stationary phase of the chromatography is octadecylsilane chemically bonded silica, a mobile phase is buffer saline solution-acetonitrile, and the related substances are selected from any one of related substances 1, 2, 3 and 4 or a combination thereof. The method can effectively separate the oxcarbazepine starting material compound of the formula II and related substances, and has good sensitivity and durability.
Figure ZY_1

Description

High performance liquid chromatography for separating and detecting oxcarbazepine starting material and related substances thereof
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a high performance liquid chromatography method for efficiently separating and detecting oxcarbazepine starting materials and related substances thereof.
Background
Oxcarbazepine (Oxcarbazepine) is an antiepileptic developed and marketed by the company nova under the chemical name: 10, 11-dihydro-10-oxo-5H-dibenzo [ b, f ] aza-5-carboxamide having the structure of formula I,
Figure SMS_1
the compounds of formula II are key starting materials for the preparation of oxcarbazepine,
Figure SMS_2
drug impurities are classified into process impurities (including reactants and reagents which are not completely reacted in the preparation, intermediates, byproducts, etc.), degradation products, impurities mixed from the reactants and reagents, etc. Is important to the impurity analysis and control of the raw material medicine starting materials. The relevant impurity conditions and separation detection methods of the starting materials used for preparing oxcarbazepine, namely the compound of the formula II are not reported. There is an urgent need in the art to develop effective methods for isolating and detecting compounds of formula II and their related impurities.
Disclosure of Invention
The invention aims at providing a high-efficiency liquid chromatography for efficiently separating and detecting oxcarbazepine key starting material compound of formula II and related substances thereof, wherein a chromatographic column stationary phase is octadecylsilane chemically bonded silica, a mobile phase is buffer saline solution-acetonitrile, and the high-efficiency liquid chromatography is matched with an ultraviolet detector.
In a preferred embodiment of the present invention, the octadecyl silane chemically bonded silica column is commercially available, preferably an octadecyl silane chemically bonded silica column manufactured by any of ThermoFisher, YMC, phenomenex, ES, merck, agilent, kromasil, agela or technate.
In a preferred technical scheme of the present invention, the octadecylsilane chemically bonded silica column is selected from Eclipse Plus C18, kromasil 100-5-C18, kromasil Eternity-5-C18, kromasil EternityXT-10-C18, kromasil 100-10-C18, kromasil 300-5-C18, YMC Triat C18, YMC ODS C18, YMC-Pack C18, phenomenex kinetex C C18, titank C18, ES-C18, epic C18, ZORBAX SB-C18, ZORBAX 300SB-C18, pogoshell 120 EC-C18, XDB-C18, TC-C18, extend-C18, bonshell ASB C18, venusil C18 Plus, venusil XBP C18 (A), venusil XBP C18 (B), venusil MP 18, innoval Neo C18, UG C18, or a combination of any of them.
In a preferred embodiment of the present invention, the octadecylsilane chemically bonded silica chromatographic column is preferably Agilent Microspher C18.
In a preferred technical scheme of the invention, the chromatographic column is specifically Agilent Microspher C, 100 mm ×4.6mm and 3 μm.
In a preferred embodiment of the present invention, the column temperature of the chromatographic column is from 23℃to 37℃and preferably from 25℃to 35℃and more preferably from 28℃to 32 ℃.
In a preferred embodiment of the present invention, the buffered saline solution is selected from any one of an ammonium formate buffered saline solution, an ammonium acetate buffered saline solution, an ammonium phosphate buffered saline solution, a potassium dihydrogen phosphate buffered saline solution, a sodium dihydrogen phosphate buffered saline solution, an ammonium dihydrogen phosphate buffered saline solution, or a combination thereof.
In a preferred embodiment of the present invention, the buffered saline solution is selected from the group consisting of monoammonium phosphate buffered saline solutions having a concentration of 1-10mg/ml, preferably 2-5 mg/ml, more preferably 2.5-3.5 mg/ml.
In the preferred technical scheme of the invention, the volume ratio of the ammonium dihydrogen phosphate buffer saline solution to acetonitrile is 70-40:30-60, preferably 65-45:35-55, more preferably 60-50:40-50.
In a preferred embodiment of the present invention, the flow rate of the mobile phase is 0.7. 0.7 ml/min-1.4. 1.4 ml/min, preferably 0.9. 0.9 ml/min-1.2. 1.2 ml/min, more preferably 0.9. 0.9 ml/min-1.1. 1.1 ml/min.
In a preferred embodiment of the present invention, the detection wavelength of the ultraviolet detector is 200 nm-240 nm, preferably 205 nm-235 nm, more preferably 210-230nm.
In a preferred embodiment of the invention, the sample volume is 10-30. Mu.l, preferably 15-25. Mu.l, more preferably 20-23. Mu.l.
In a preferred embodiment of the present invention, the substance of interest is selected from any one of the substances of interest 1, 2, 3, 4 or a combination thereof,
Figure SMS_3
。
in a preferred embodiment of the present invention, the peak-exiting sequence is related substance 1, related substance 2, related substance 3, related substance 4, formulas II-1 and II-2 in order, wherein the separation degree of each related substance peak and the compound peak of formula II from the adjacent compound peak is greater than 1.5.
In a preferred embodiment of the present invention, the high performance liquid chromatography comprises the steps of:
1) Preparing a solution:
solvent: acetonitrile-water (70:30);
positioning solution: taking appropriate amounts of reference substances of the formulas II-1, II-2, related substances 1, 2, 3 and 4, dissolving with a solvent, and diluting to obtain single positioning solutions with the concentration of 100 mug/ml;
mixing solution: taking a proper amount of each of the reference substances of the formulas II-1, II-2, related substances 1, 2, 3 and 4, dissolving and diluting the reference substances with a solvent to prepare a mixed solution with the concentration of about 5 mug/ml;
test solution: taking a proper amount of oxcarbazepine starting materials, and preparing a solution with the concentration of about 1mg/ml by using a solvent;
2) Octadecylsilane chemically bonded silica chromatographic column Agilent MicroSpher C, 100×4.6mm and 3 μm is used as stationary phase, the column temperature is 28-32 ℃, the mobile phase is monoammonium phosphate buffer saline solution (3.2 g monoammonium phosphate is taken and 1000ml water is added for dissolution) -acetonitrile (57-53:43-47), the flow rate is 0.9ml/min-1.1 ml/min, the detection wavelength of an ultraviolet detector is 210nm, the sample injection amount is 20 μl, and a chromatogram is recorded.
In a preferred embodiment of the present invention, the high performance liquid chromatography comprises the steps of:
1) Preparing a solution:
solvent: acetonitrile-water (70:30);
positioning solution: taking appropriate amounts of reference substances of the formulas II-1, II-2, related substances 1, 2, 3 and 4, dissolving with a solvent, and diluting to obtain single positioning solutions with the concentration of 100 mug/ml;
mixing solution: taking a proper amount of each of the reference substances of the formulas II-1, II-2, related substances 1, 2, 3 and 4, dissolving and diluting the reference substances with a solvent to prepare a mixed solution with the concentration of about 5 mug/ml;
test solution: taking a proper amount of oxcarbazepine starting materials, and preparing a solution with the concentration of about 1mg/ml by using a solvent;
2) Octadecylsilane chemically bonded silica chromatographic column Agilent MicroSpher C, 100×4.6mm,3 μm as stationary phase, column temperature of 30deg.C, mobile phase of monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water added for dissolution) -acetonitrile (55:45), flow rate of 1.0ml/min, detection wavelength of ultraviolet detector of 210nm, sample injection amount of 20 μl, and record chromatogram.
It is another object of the present invention to provide the use of the high performance liquid chromatography for separating and detecting oxcarbazepine key starting material compounds of formula II and related substances thereof according to the present invention for the efficient separation and detection of oxcarbazepine key starting material compounds of formula II and related substances thereof.
Unless otherwise indicated, when the invention relates to a percentage between liquids, the percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentage between solids and liquids, the percentage being weight/volume percentage; the balance being weight/weight percent.
Compared with the prior art, the invention has the following beneficial effects: the invention optimizes the elution condition and detection condition of high performance liquid chromatography through scientific screening, realizes the effective separation and detection of 6 compounds which have similar structures and are difficult to separate in total and are the key starting material formula II of oxcarbazepine and related substances thereof, has good separation effect, realizes the quality control of the key starting material formula II of oxcarbazepine and the preparation medicine (oxcarbazepine) thereof, obviously improves the quality of the preparation medicine, reduces adverse reactions possibly occurring in clinical experiments and treatment of the medicine, and ensures the effectiveness and safety of the medicine administration of patients.
Drawings
Fig. 1 is a high performance liquid chromatogram of comparative example 1.
FIG. 2 is a high performance liquid chromatogram of comparative example 2.
FIG. 3 is a high performance liquid chromatogram of comparative example 3.
FIG. 4 is a high performance liquid chromatogram of comparative example 4.
FIG. 5 is a high performance liquid chromatogram of comparative example 5.
FIG. 6 is a high performance liquid chromatogram of comparative example 6.
FIG. 7 is a high performance liquid chromatogram of comparative example 7.
FIG. 8 is a high performance liquid chromatogram of the mixed solution of comparative example 8.
FIG. 9 is a high performance liquid chromatogram of the sample solution of comparative example 8.
FIG. 10 is a high performance liquid chromatogram of comparative example 9.
Fig. 11 is a high performance liquid chromatogram of example 1.
FIG. 12 is a high performance liquid chromatogram of the mixed solution of example 3.
FIG. 13 is a high performance liquid chromatogram of the sample solution of example 4.
FIG. 14 is a high performance liquid chromatogram of the positioning solution of substance 4 of example 4.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail hereinafter with reference to the accompanying drawings. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be arbitrarily combined with each other.
Comparative example 1 HPLC separation detection of Compound of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: kromasil C18, 250 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile-isopropanol (50:25:25);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (1:1);
sample injection amount: 20 μl;
mixing solution: appropriate amounts of each of the formula II-1, the formula II-2, the related substance 1, the related substance 2 and the related substance 3 were prepared into a mixed solution (each concentration was about 0.1 mg/ml) with a solvent.
The chromatographic results are shown in FIG. 1. The results show that: the middle 3 chromatographic peaks of the 5 chromatographic peaks do not reach baseline separation, cannot be effectively separated and detected, and cannot be used for controlling the product quality.
Comparative example 2 HPLC separation detection of Compound of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: kromasil C18, 250 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase a: ammonium dihydrogen phosphate buffer saline solution (taking ammonium dihydrogen phosphate 3.2g, adding water 1000ml for dissolution);
mobile phase B: acetonitrile-isopropanol (1:1);
mobile phase a-mobile phase B (55:45);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (1:1);
sample injection amount: 20 μl;
mixing solution: appropriate amounts of each of the formula II-1, the formula II-2, the related substance 1, the related substance 2 and the related substance 3 were prepared into a mixed solution (each concentration was about 0.1 mg/ml) with a solvent.
The chromatographic results are shown in FIG. 2. The results show that: 2 chromatographic peaks in the 5 chromatographic peaks are overlapped, so that the separation and detection cannot be effectively performed, and the quality of a product cannot be controlled.
Comparative example 3 HPLC separation detection of Compound of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: kromasil C18, 250 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (55:45);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (1:1);
sample injection amount: 20 μl;
mixing solution: appropriate amounts of each of the formula II-1, the formula II-2, the related substance 1, the related substance 2 and the related substance 3 were prepared into a mixed solution (each concentration was about 0.1 mg/ml) with a solvent.
The chromatographic results are shown in FIG. 3. The results show that: the middle 3 chromatographic peaks did not reach baseline separation and could not be used to control product quality.
Comparative example 4 HPLC separation detection of Compounds of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: kromasil C18, 250 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase a: ammonium dihydrogen phosphate buffer saline solution (taking ammonium dihydrogen phosphate 3.2g, adding water 1000ml for dissolution);
mobile phase B: acetonitrile;
flow rate: 1.0ml/min;
gradient elution procedure:
Figure SMS_4
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (1:1);
sample injection amount: 20 μl;
mixing solution: appropriate amounts of each of the formula II-1, the formula II-2, the related substance 1, the related substance 2 and the related substance 3 were prepared into a mixed solution (each concentration was about 0.1 mg/ml) with a solvent.
The chromatographic results are shown in FIG. 4. The results show that: the middle three peaks are still not effectively separated and cannot be used to control product quality.
Comparative example 5 HPLC separation detection of Compound of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: warters XTERRA PR18, 250 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (55:45);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (1:1);
sample injection amount: 20 μl;
mixing solution: appropriate amounts of each of the formula II-1, the formula II-2, the related substance 1, the related substance 2 and the related substance 3 were prepared as mixed solutions (each concentration was about 0.1 mg/ml) with a solvent.
The chromatographic results are shown in FIG. 5. The results show that: 2 chromatographic peaks in the 5 chromatographic peaks are completely overlapped, so that the separation and detection can not be effectively carried out, and the product quality can not be controlled.
Comparative example 6 HPLC separation detection of Compound of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: warters XTERRA PR18, 250 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (48:52);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (1:1);
sample injection amount: 20 μl;
mixing solution: appropriate amounts of each of the formula II-1, the formula II-2, the related substance 1, the related substance 2 and the related substance 3 were prepared into a mixed solution (each concentration was about 0.1 mg/ml) with a solvent.
The chromatographic results are shown in Table 1 below and FIG. 6. The results show that: 5 chromatographic peaks can be detected, but the minimum separation degree is 1.47, so that the detection can not be effectively separated and the product quality can not be controlled.
Figure SMS_5
Comparative example 7 HPLC separation detection of Compound of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: agilent ZoRBAX SB-C18, 150 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (48:52);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (1:1);
sample injection amount: 20 μl;
mixing solution: appropriate amounts of each of the formula II-1, the formula II-2, the related substance 1, the related substance 2 and the related substance 3 were prepared into a mixed solution (each concentration was about 0.1 mg/ml) with a solvent.
The chromatographic results are shown in Table 2 below and FIG. 7. The results show that: 5 chromatographic peaks can be detected, but the minimum separation degree is 1.35, so that the detection can not be effectively separated and the product quality can not be controlled.
Figure SMS_6
Comparative example 8 HPLC separation detection of Compound of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: agilent ZoRBAX SB-C18, 150 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (52:48);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (70:30);
sample injection amount: 20 μl;
mixing solution: appropriate amounts of each of the formula II-1, the formula II-2, the related substance 1, the related substance 2 and the related substance 3 were prepared into a mixed solution (each concentration was about 0.1 mg/ml) with a solvent.
The chromatographic results are shown in FIG. 8. The results show that: the mixed solution was able to detect 5 chromatographic peaks with a minimum degree of separation of 1.65.
The chromatographic conditions are selected for separation and detection of the sample solution.
Test solution: the oxcarbazepine starting material was prepared in a suitable amount and with a solvent to a concentration of about 1 mg/ml.
The chromatographic results of the test sample solution are shown in FIG. 9. The results show that: the main peak 1 in the sample solution cannot be separated from the previous unknown impurity peak.
Comparative example 9 HPLC separation detection of Compound of formula II and related substances
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: agilent ZoRBAX SB-C18, 150 mm. Times.4.6 mm,5 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (55:45);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (70:30);
sample injection amount: 20 μl;
test solution: the oxcarbazepine starting material was prepared in a suitable amount and with a solvent to a concentration of about 1 mg/ml.
The chromatographic results are shown in FIG. 10. The results show that: the main peak 1 in the sample solution is still not effectively separated from the previous unknown impurity peak.
EXAMPLE 1 HPLC method for the separation and detection of Compounds of formula II and related substances according to the invention
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: agilent MicroSpher C18, 100×4.6mm,3 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (55:45);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (70:30);
sample injection amount: 20 μl;
test solution: the oxcarbazepine starting material was prepared in a suitable amount and with a solvent to a concentration of about 1 mg/ml.
The results are shown in Table 3 and chromatogram 11. The results show that: the separation degree of the main peak 1 and the previous unknown impurity peak in the sample meets the requirement.
Figure SMS_7
Separating and structural identifying the unknown impurity: m/z (ESI) + ) 335.8; 1 H-NMR（400MHz,CDCl 3 ）7.29~7.43（m,8H）, 5.40~5.98（m,1H）, 3.98~4.12（m,1H）, 3.29~3.57（m1H), the structure was resolved into the related substance 4 of the present invention.
EXAMPLE 2 preparation of substance 4 according to the present invention
To a 100ml three-necked flask, 20ml of chlorobenzene and 5.0g of the relevant substance 2 were added, the temperature was raised to 75℃and a chlorobenzene solution of bromine (bromine 1.5g was dissolved in 20ml of chlorobenzene) was added dropwise, the progress of the reaction was monitored by TLC (developing agent: PE/EA=8/1), and after the completion of the reaction, the reaction was cooled to room temperature, and the reaction solution was washed with 50ml of water and concentrated to dryness under reduced pressure to give a yellowish brown oil. Column chromatography gave 0.38g of white solid using n-hexane/ethyl acetate=20/1 as eluent.
EXAMPLE 3 specificity experiments of the method of the invention
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: agilent MicroSpher C18, 100×4.6mm,3 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (55:45);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (70:30);
sample injection amount: 20 μl;
positioning solution: taking appropriate amounts of reference substances of the formulas II-1, II-2, related substances 1, 2, 3 and 4, dissolving and diluting with a solvent to prepare single positioning solutions with the concentration of 100 mug/ml;
mixing solution: and (3) taking a proper amount of each of the reference substances of the formulas II-1 and II-2, the related substance 1, the related substance 2, the related substance 3 and the related substance 4, dissolving the reference substances by using a solvent, and diluting the reference substances to prepare a mixed solution with the concentration of about 5 mug/ml. And taking 20 mu l of each of the mixed solution and the single positioning solution, and injecting the mixed solution and the single positioning solution into a high performance liquid chromatograph.
The mixed solution results are shown in Table 4 below and chromatogram 12.
Figure SMS_8
The results show that: under the method, the separation degree between the impurities and the main component peak meets the requirement, and the specificity is good.
Example 4 HPLC separation detection of test solution
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: agilent MicroSpher C18, 100×4.6mm,3 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (55:45);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (70:30);
sample injection amount: 20 μl;
test solution: taking a proper amount of oxcarbazepine starting materials, and preparing a solution with the concentration of about 1mg/ml by using a solvent;
substance 4 positioning solution: and taking a proper amount of the reference substance of the related substance 4, dissolving the reference substance by using a solvent, and diluting the reference substance to prepare a positioning solution with the concentration of about 0.2 mg/ml.
The test solution results are shown in Table 5 and chromatogram 13 below, and the relevant substance 4 positioning solution chromatogram results are shown in FIG. 14. Further, it was confirmed that the impurity peak preceding the main peak 1 in the sample solution was related substance 4.
Figure SMS_9
Example 5 sensitivity experiments of the method of the invention
Liquid chromatograph: shimadzu LC-20A;
chromatographic column: agilent MicroSpher C18, 100×4.6mm,3 μm;
column temperature: 30 ℃;
mobile phase: monoammonium phosphate buffer saline solution (monoammonium phosphate 3.2g, 1000ml of water is added for dissolution) -acetonitrile (55:45);
flow rate: 1.0ml/min;
a detector: an ultraviolet detector;
detection wavelength: 210nm;
solvent: acetonitrile-water (70:30);
sample injection amount: 20 μl;
preparing a solution: and (3) taking a proper amount of each of the reference substances of the formulas II-1, II-2, related substances 1, 2, related substances 3 and related substances 4, dissolving and diluting the reference substances by using a solvent to prepare a mixed solution with the concentration of about 5 mug/ml, gradually diluting the solution by using the solvent, taking the solution with the signal to noise ratio of about 10 as a quantitative limit solution, and taking the solution with the signal to noise ratio of about 3 as a detection limit solution.
And (3) taking 20 mu l of each solution, injecting the solution into a high performance liquid chromatograph, and determining according to a law, wherein the test results are shown in the table 6 below.
Figure SMS_10
The results show that: the method for detecting each impurity has high sensitivity, and can finish quantitative detection when each compound is low to ng level.
EXAMPLE 6 durability test of the method of the invention
Test solution: the oxcarbazepine starting material was taken in an appropriate amount, dissolved in a solvent and diluted to a solution having a concentration of about 1mg/ml, which was injected into a high performance liquid chromatograph and measured by the method, and the test results are shown in Table 7 below.
Figure SMS_11
The results show that: the column temperature is 30+/-2 ℃, the flow rate is 1.0+/-0.1 ml/min, the acetonitrile proportion in the mobile phase is changed within the range of 45+/-2%, the separation degree between related substances 1-4 and the separation degree between the related substances and the main component are all in accordance with the requirements, the detection amount of impurities in the sample is basically consistent, the chromatographic condition has no influence on the measurement result within the change range, and the method has good durability.
Although the embodiments disclosed in the present invention are described above, the embodiments are merely used to facilitate understanding of the present invention, and are not intended to limit the present invention. Any person skilled in the art can make any modification and variation in form and detail without departing from the spirit and scope of the present disclosure, but the scope of the present disclosure is to be determined by the appended claims.

Claims (3)

1. A high performance liquid chromatography method for efficiently separating and detecting oxcarbazepine key starting materials and related substances, wherein a chromatographic column stationary phase is Agilent Microspher C, 100 mm ×4.6mm,3 [ mu ] m, a chromatographic column temperature is 28 ℃ -32 ℃, a mobile phase is monoammonium phosphate buffer saline-acetonitrile, the concentration of the monoammonium phosphate buffer saline is 3.2 mg/ml, and the volume ratio of the monoammonium phosphate buffer saline to acetonitrile is 57-53:43-47, the flow rate of the mobile phase is 0.9-ml-1.1 ml/min, the detection wavelength of the ultraviolet detector is 210nm, the sample injection volume is 20 μl, the starting materials are compounds of formula II-1 and formula II-2,
Figure QLYQS_1
，
wherein, when R is Cl, the compound is shown as a formula II-1, when R is Br, the compound is shown as a formula II-2,
the related substances are selected from related substances 1, 2, 3 and 4,
Figure QLYQS_2
2. the high performance liquid chromatography according to claim 1, wherein the peak order of the compounds of formula II-1, II-2 and related substances is related substance 1, related substance 2, related substance 3, related substance 4, formula II-1 and II-2, wherein the separation degree of each related substance peak and the peaks of the compounds of formula II-1, II-2 from the peaks of the adjacent compounds is greater than 1.5.
3. The high performance liquid chromatography of any one of claims 1-2, comprising the steps of:
1) Preparing a solution:
solvent: acetonitrile-water, wherein the volume ratio of acetonitrile to water is 70:30;
positioning solution: taking appropriate amounts of reference substances of the formulas II-1, II-2, related substances 1, 2, 3 and 4, dissolving with a solvent, and diluting to obtain single positioning solutions with the concentration of 100 mug/ml;
mixing solution: taking a proper amount of each of the reference substances of the formulas II-1, II-2, related substances 1, 2, 3 and 4, dissolving and diluting the reference substances with a solvent to prepare a mixed solution with the concentration of about 5 mug/ml;
test solution: taking a proper amount of oxcarbazepine starting materials, and preparing a solution with the concentration of about 1mg/ml by using a solvent;
2) The chromatographic column Agilent MicroSpher C, 100 mm ×4.6mm and 3 μm are taken as stationary phase, the column temperature is 30 ℃, the mobile phase is monoammonium phosphate buffer saline solution-acetonitrile, the preparation method of the monoammonium phosphate buffer saline solution is that 3.2g monoammonium phosphate is taken and 1000ml water is added to dissolve the monoammonium phosphate buffer saline solution, the volume ratio of the monoammonium phosphate buffer saline solution to acetonitrile is 55:45, the flow rate is 1.0ml/min, the detection wavelength of an ultraviolet detector is 210nm, the sample injection amount is 20 μl, and the elution separation and the chromatogram are recorded.
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