CN116626209B - High-sensitivity oxcarbazepine starting material content detection method and application thereof - Google Patents
High-sensitivity oxcarbazepine starting material content detection method and application thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 40
- CTRLABGOLIVAIY-UHFFFAOYSA-N oxcarbazepine Chemical compound C1C(=O)C2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 CTRLABGOLIVAIY-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 229960001816 oxcarbazepine Drugs 0.000 title claims abstract description 28
- 239000007858 starting material Substances 0.000 title claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims abstract description 49
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 21
- 238000005173 quadrupole mass spectroscopy Methods 0.000 claims abstract description 17
- 238000010828 elution Methods 0.000 claims abstract description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 11
- 238000012544 monitoring process Methods 0.000 claims abstract description 8
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 7
- 230000035945 sensitivity Effects 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 5
- 238000000132 electrospray ionisation Methods 0.000 claims abstract description 5
- 238000010812 external standard method Methods 0.000 claims abstract description 5
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims abstract description 5
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 3
- 239000000741 silica gel Substances 0.000 claims abstract description 3
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 72
- 239000000243 solution Substances 0.000 claims description 29
- 239000000523 sample Substances 0.000 claims description 21
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 18
- 238000005303 weighing Methods 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 14
- 239000012535 impurity Substances 0.000 claims description 14
- 238000004811 liquid chromatography Methods 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 10
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical group COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 9
- 235000019253 formic acid Nutrition 0.000 claims description 9
- 239000012488 sample solution Substances 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 7
- 238000001819 mass spectrum Methods 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 239000013558 reference substance Substances 0.000 claims description 6
- 239000012085 test solution Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 238000005507 spraying Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 claims description 4
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 3
- 239000012088 reference solution Substances 0.000 claims description 3
- 150000002500 ions Chemical class 0.000 claims 6
- 238000004090 dissolution Methods 0.000 claims 1
- 239000002808 molecular sieve Substances 0.000 claims 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract 1
- 239000011259 mixed solution Substances 0.000 description 10
- 239000002904 solvent Substances 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 8
- 238000010586 diagram Methods 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000003814 drug Substances 0.000 description 4
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 3
- 241001251200 Agelas Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 231100000024 genotoxic Toxicity 0.000 description 2
- 230000001738 genotoxic effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 1
- 150000001266 acyl halides Chemical class 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 125000001188 haloalkyl group Chemical group 0.000 description 1
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- 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
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- 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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- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- 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
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- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- 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
- G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
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Abstract
The invention provides a high-sensitivity oxcarbazepine initial material content detection method and application thereof, wherein the detection method is high-performance liquid chromatography-triple quadrupole mass spectrometry combined detection, a chromatographic column stationary phase of the high-performance liquid chromatography is octadecylsilane chemically bonded silica gel, a mobile phase adopts a gradient elution mode, an electrospray ionization source is used as an ion source in mass spectrometry detection, a multi-reaction monitoring mode is adopted, and a sample is injected into a high-performance liquid chromatography-mass spectrometry combined instrument to obtain the content according to an external standard method by peak area calculation. The method can detect the content of the compound shown as the formula II of the oxcarbazepine key starting material with high sensitivity, and effectively ensures the quality of the oxcarbazepine finally prepared.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a high-sensitivity oxcarbazepine starting material content detection method and application 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,
the compounds of formula II are key starting materials for the preparation of oxcarbazepine,
the oxcarbazepine preparation process is as follows:
。
according to the rule of ICH M7 guidelines, the formula II-1 and the formula II-2 are potential genotoxic compounds with acyl halide and halogenoalkyl warning structures, and the risk of bringing the compounds into the final product along with the reaction is caused, so that strict content control is required for the two compounds to ensure the safety of the oxcarbazepine finished product. The limits of formula II-1 and formula II-2 in the final oxcarbazepine product were formulated to be about 0.35 ppm, respectively, based on the human genotoxic substance threshold (TTC) and oxcarbazepine maximum daily dose (4.2 g) conversion. The sensitivity level cannot be achieved by the common high performance liquid chromatography, and there is an urgent need in the art to develop a content detection method which is suitable for more than two compounds and can meet the sensitivity requirement.
Disclosure of Invention
The invention aims at providing a method for detecting the content of an oxcarbazepine key starting material compound shown as a formula II, wherein the method is high performance liquid chromatography-triple quadrupole mass spectrometry combined detection, and 1) the conditions of the high performance liquid chromatography are as follows: the chromatographic column stationary phase is octadecylsilane chemically bonded silica gel, the mobile phase is composed of A phase and B phase, the A phase is formic acid aqueous solution, the B phase is acetonitrile aqueous solution, the elution condition is gradient elution, the column temperature of the chromatographic column is 35-45 ℃, the flow rate of the mobile phase is 0.1-1mL/min, the sample injection amount is 1-7 mu L, and 2) the conditions of mass spectrum are: the electrospray ionization source (ESI source) is used as an ion source, the monitoring mode is multi-reaction monitoring (MRM), the mass spectrum detection mode is positive ion mode, the gas curtain gas pressure is 25-35psi, the ionization voltage is 5500V, the temperature is 550 ℃, the spraying collision gas pressure is 7-11psi, the spraying gas pressure is 50-60psi, the auxiliary heating gas pressure is 50-60psi, and 3) the measuring method comprises the following steps: preparing reference solution and sample solution respectively, injecting into high performance liquid chromatography-mass spectrometer, recording MRM, and calculating according to external standard method and peak area.
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 C18, titank C18, ES-C18, epic C18, ZORBAX SB-C18, ZORBAX 300SB-C18, pogoshel 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, XD C18, UG 18, 35C 18-75 mat II, or any combination thereof.
In a preferred embodiment of the invention, the octadecylsilane chemically bonded silica column is Infinitylab poroshell, 120, EC to C18,3mm×100mm,1.9 μm.
In a preferred embodiment of the present invention, the column temperature of the chromatographic column is preferably 37 ℃ to 43 ℃.
In a preferred embodiment of the present invention, the flow rate of the mobile phase is preferably 0.1-0.6 mL/min.
In a preferred embodiment of the present invention, the sample injection amount is preferably 1-3 μl.
In a preferred technical scheme of the invention, the volume fraction of formic acid in the phase A formic acid aqueous solution is 0.1% -2%, preferably 0.1% -0.5%.
In a preferred technical scheme of the invention, the volume fraction of acetonitrile in the phase B acetonitrile aqueous solution is 90% -99%, preferably 93% -97%.
In a preferred technical scheme of the invention, the gradient elution procedure is as follows:
。
in a preferred embodiment of the present invention, the air curtain has an air pressure of 28-32psi.
In a preferred embodiment of the present invention, the impinging air pressure is 8-10psi.
In a preferred embodiment of the invention, the spray gas pressure is 53-57psi.
In a preferred embodiment of the present invention, the auxiliary heating gas pressure is 53-57psi.
In a preferred embodiment of the invention, the mass spectrometric detection ion pairs of the compound of formula II are:
。
in the preferred technical scheme of the invention, the valve switching time in mass spectrum detection is as follows:
。
in the preferred technical scheme of the invention, the high performance liquid chromatography-mass spectrometry combined detection method specifically comprises the following steps:
(1) liquid chromatography conditions:
chromatographic column: infinitylab poroshell 120 EC-C18,3mm×100mm,1.9 μm
Mobile phase: phase A is 0.1% formic acid aqueous solution, phase B is 95% acetonitrile aqueous solution
Flow rate: 0.3mL/min
Column temperature: 40 DEG C
Sample injection amount: 1 mu L
Gradient elution conditions:
，
(2) mass spectrometry conditions:
ion source: electrospray ionization source (ESI source)
Monitoring mode: multiple Reaction Monitoring (MRM) mode
Detection mode: positive ion mode
Air curtain air pressure: 30psi
Ionization voltage of 5500V
The temperature is 550 DEG C
The impinging air pressure was 9psi
The spray gas pressure was 55psi
The auxiliary heating air pressure is 55psi
The ion pairs are:
，
valve switching time:
，
(3) the measuring method comprises the following steps:
control solution: taking a proper amount of impurity compound reference substances of the formulas II-1 and II-2, precisely weighing, adding acetonitrile for dissolving and diluting to prepare solutions with about 17.5ng each 1 mL;
test solution: precisely weighing 500mg of the sample, placing in a 10mL measuring flask, adding acetonitrile for dissolving and diluting to a scale, and shaking for later use;
precisely measuring 1 μl of each of the sample solution and the reference solution, respectively injecting into high performance liquid chromatography-mass spectrometry detector according to the detection conditions, recording MRM, and calculating with peak area according to external standard method.
The invention also aims to provide the application of the method for detecting the content of the compound shown as the formula II of the oxcarbazepine key starting material in the method for detecting the content of the compound shown as the formula II of the oxcarbazepine key starting material with high sensitivity.
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 method can detect the content of the oxcarbazepine key starting material formula II compound with high sensitivity, effectively realize the quality control of the oxcarbazepine finally prepared, obviously improve the quality of the medicines, reduce adverse reactions possibly occurring in the treatment of the medicines and ensure the safety and the effectiveness of the medication of patients.
Drawings
FIG. 1 is a characteristic ion flow diagram of the compound of formula II-1 of example 1.
FIG. 2 is a characteristic ion flow diagram of the compound of formula II-2 of example 1.
Fig. 3 is a characteristic ion flow diagram of oxcarbazepine of example 2.
FIG. 4 is a characteristic ion flow diagram of the compound of formula II-1 of example 2.
FIG. 5 is a characteristic ion flow diagram of the compound of formula II-2 of example 2.
Fig. 6 is a characteristic ion-flow diagram of the compound of formula III of example 6.
FIG. 7 is a characteristic ion flow diagram of the compound of formula II-1 of example 6.
FIG. 8 is a characteristic ion flow diagram of the compound of formula II-2 of example 6.
FIG. 9 is a linear plot of the compound of formula II-1 of example 8.
FIG. 10 is a linear plot of the compound of formula II-2 of example 8.
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.
Example 1 high performance liquid chromatography-triple quadrupole mass spectrometry combination detection of Compounds of formulas II-1 and II-2
1) Conditions of liquid chromatography
Chromatographic column: infinitylab poroshell 120-EC-C18 (3 mm. Times.100 mm,1.9 μm),
mobile phase: phase A is 0.1% formic acid aqueous solution, phase B is 95% acetonitrile aqueous solution,
the flow rate is 0.3mL/min, the column temperature is 40 ℃, the sample injection amount is 1 mu L,
preparing a mixed solution: weighing appropriate amounts of oxcarbazepine, II-1 and II-2, preparing into mixed solution with concentration of about 200ng/ml with acetonitrile,
gradient elution procedure is as follows table 1:
，
2) Mass spectrometry conditions
Ion source: the source of the ESI is provided by,
monitoring mode: multiple Reaction Monitoring (MRM),
detection mode: a positive ion mode of operation, in which,
the air curtain air pressure was 30psi,
the ionization voltage is 5500V,
the temperature was 550 c,
the impinging air pressure was 9psi,
the spray gas pressure was 55psi,
the auxiliary heating gas pressure was 55psi,
ion pairs are shown in table 2 below:
。
the results show that: the peak time of the compounds of the formulas II-1 and II-2 is between 9 and 10min (shown in figures 1-2), the peak shape is good, and the elution condition is optimized to lead the peak time of the two impurities to be advanced.
Example 2 high Performance liquid chromatography-triple quadrupole Mass Spectrometry combination detection of Compounds of formulas II-1 and II-2
1) Conditions of liquid chromatography
Gradient elution procedure as in table 3 below, the remaining conditions were the same as in example 1,
，
2) Mass spectrometry conditions
The conditions were the same as in example 1.
The results show that: the main peak time of oxcarbazepine is 1.69min, the peak time of II-1 is 4.69min, the peak time of II-2 is 5.02min (figure 3-5), the separation degree meets the requirement of cutting the test sample into the waste liquid in the test sample solution with limited concentration, and the concentration and the solubility of the test sample are confirmed according to the response conditions of II-1 and II-2.
Example 3 high Performance liquid chromatography-triple quadrupole Mass Spectrometry combination detection of Compounds of formulas II-1 and II-2
1) Conditions of liquid chromatography
The solvents of the mixed solution are respectively as follows: DMSO, DMF or acetonitrile (as oxcarbazepine of the test sample satisfying the limit concentration of impurities is not completely dissolved in acetonitrile, the other two solvents were examined), the other conditions were the same as in example 2,
2) Mass spectrometry conditions
The conditions were the same as in example 2.
The results show that: the responses of the impurities in the three solvents were compared and the signal to noise ratio was acetonitrile > DMF > DMSO, therefore DMF was used as solvent.
Example 4 high performance liquid chromatography-triple quadrupole mass spectrometry combination detection of Compounds of formulas II-1 and II-2
1) Conditions of liquid chromatography
Preparing a solution:
determining the limit concentration of the impurity to be 17.5ng/ml according to the response and limit of the impurity, and determining the concentration of the test sample to be 50mg/ml;
test solution: weighing a proper amount of oxcarbazepine, and preparing into a solution with the concentration of 50mg/ml by using DMF;
limited concentration mixed solution: weighing a proper amount of each of the compounds of the formulas II-1 and II-2, and preparing a solution with the concentration of 17.5ng/ml by using DMF;
sample solution containing a limited concentration of impurities: respectively weighing oxcarbazepine, a compound of the formula II-1 and a compound of the formula II-2, and preparing into solutions with the concentration of 50mg/ml, 17.5ng/ml and 17.5ng/ml respectively by using DMF;
the other conditions were the same as in example 2,
2) Mass spectrometry conditions
The conditions were the same as in example 2.
The results show that: the solution stability experiment for 12 hours shows that the formula II-2 is unstable under the condition of DMF as a solvent, acetonitrile is continuously adopted as the solvent in the next step, and the content of two impurity compounds is detected in the sample of the formula III.
Example 5 high performance liquid chromatography-triple quadrupole mass spectrometry combination detection of Compounds of formulas II-1 and II-2
1) Conditions of liquid chromatography
Weighing appropriate amounts of formula III, II-1 and II-2, preparing into mixed solution with acetonitrile and concentration of about 200ng/ml, and the rest conditions are the same as in example 4,
2) Mass spectrometry conditions
Ion pairs are shown in Table 4 below, with the remaining conditions being the same as in example 2,
。
the results show that: the peak times of the compounds III, II-1 and II-2 were 3.48min,4.19min and 4.55min, respectively, and the solution stability experiment for 12 hours showed that the solution was stable for 12 hours.
Comparative example 1 high performance liquid chromatography-triple quadrupole mass spectrometry combination detection of compounds of formulas II-1 and II-2
1) Conditions of liquid chromatography
Chromatographic column: agela Venusil HILIC (2.1X100 mm, 5 μm);
the flow rate was 0.6ml/min,
gradient elution procedure is as follows table 5:
，
the other conditions were the same as in example 5,
2) Mass spectrometry conditions
In the same manner as in example 5,
the results show that: only the compound of formula III showed a peak, neither of formulas II-1 nor II-2 showed a peak,
the gradient elution procedure was adjusted as follows in table 6:
the results show that: the compounds of formula II-1 and II-2 still have no peaks.
EXAMPLE 6 high Performance liquid chromatography-triple quadrupole Mass Spectrometry (HPLC-triple quadrupole Mass Spectrometry) detection of Compounds of formulas II-1 and II-2
1) Conditions of liquid chromatography
Preparing a solution:
test solution: weighing a proper amount of a compound of the formula III, and preparing a solution with the concentration of 50mg/ml by acetonitrile;
limited concentration mixed solution: weighing appropriate amounts of the compounds of the formulas II-1 and II-2, and preparing solutions with the concentrations of about 17.5ng/ml by acetonitrile;
sample solution containing a limited concentration of impurities: weighing appropriate amounts of the compounds of the formula III, the formula II-1 and the formula II-2 respectively, and preparing solutions with the concentrations of 50mg/ml, 17.5ng/ml and 17.5ng/ml respectively by acetonitrile;
the other conditions were the same as in example 5,
respectively sucking 1 μl of each of the above three solutions, injecting into LC-MS/MS liquid chromatography-mass spectrometer, comparing peak area values,
2) Mass spectrometry conditions
In the same manner as in example 5,
valve switching is shown in table 7 below:
。
results: under the conditions of the chromatograph and the mass spectrum, the solvent and the test sample have no interference on the determination of the impurity formulas II-1 and II-2, and the method has good specificity. Wherein the peak conditions of the compounds of formula III and II-1 and II-2 in the test solution containing the impurities with limited concentrations are shown in figures 6-8 respectively.
Example 7 high Performance liquid chromatography-triple quadrupole Mass Spectrometry (HPLC-triple quadrupole Mass Spectrometry) detection sensitivity for Compounds of formulas II-1 and II-2
The liquid chromatography conditions and mass spectrometry conditions were the same as in example 6, the limiting concentration mixed solution was precisely measured, diluted stepwise, 1. Mu.L was precisely measured, and the mixture was injected into an LC-MS/MS liquid chromatograph/mass spectrometer, and the chromatogram was recorded and analyzed. At signal to noise ratios of approximately 10:1 and 3: the solution at 1 is used as a quantitative limit and a detection limit solution, the quantitative limit and the detection limit are calculated, and the result is shown in the following table 8:
results: the detection method can meet the high-sensitivity detection requirements of the compounds of the formulas II-1 and II-2.
Example 8 high Performance liquid chromatography-triple quadrupole Mass Spectrometry (HPLC-triple quadrupole Mass Spectrometry) for detecting Linear Compounds of formulas II-1 and II-2
And (3) weighing a proper amount of mixed solution with the limit concentration, and adding a solvent to dilute the mixed solution to prepare a solution with the concentration of 25%, 50%, 75%, 100%, 150% and 250% of the limit concentration, so as to obtain a linear solution. 1 mu L of the linear solution is precisely measured, injected into an LC-MS/MS liquid chromatography-mass spectrometer and experimental data are recorded. Linear regression analysis was performed with concentrations on the abscissa and peak areas on the ordinate, with the results shown in table 9 below and in fig. 9-10:
the results show that: the concentrations and peak areas of the formulas II-1 and II-2 are in good linear relation in the range of 4.48 ng/mL-44.75 ng/mL and 4.71 ng/mL-47.1 ng/mL.
Example 9 high Performance liquid chromatography-triple quadrupole Mass Spectrometry (HPLC-triple quadrupole Mass Spectrometry) detection accuracy for Compounds of formulas II-1 and II-2
And (3) taking a proper amount of a test sample, precisely weighing, adding a proper amount of a mixed solution with a limited concentration, dissolving by using a solvent, quantitatively diluting to prepare parallel samples with the impurity concentration of 50%, 100% and 150% of the limited concentration level respectively, and preparing the parallel samples as accurate sample solutions by the same method. 1 mu L of each level of accuracy solution is precisely measured, and is respectively injected into an LC-MS/MS liquid chromatograph-mass spectrometer, and the recovery rate is calculated, and the result is shown in the following table 10:
the results show that: the average recovery rate of the method for the samples containing the formula II-1 and the formula II-2 under different concentrations is 96.88% and 107.34%, respectively, the RSD values are less than 10%, and the detection accuracy of the method is high.
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. The method for detecting the content of the oxcarbazepine key starting material compound of formula II comprises the following steps of 1) high performance liquid chromatography-triple quadrupole mass spectrometry combination detection: the chromatographic column stationary phase is octadecylsilane chemically bonded silica gel, the mobile phase is composed of A phase and B phase, the A phase is formic acid aqueous solution, the B phase is acetonitrile aqueous solution, the volume fraction of formic acid in the A phase formic acid aqueous solution is 0.1%, the volume fraction of acetonitrile in the B phase acetonitrile aqueous solution is 95%, the elution condition is gradient elution, the column temperature of the chromatographic column is 40 ℃, the flow rate of the mobile phase is 0.3mL/min, the sample injection amount is 1 mu L, and 2) the mass spectrum conditions are: the electrospray ionization source is used as an ion source, the monitoring mode is multi-reaction monitoring, the mass spectrum detection mode is positive ion mode, the air curtain air pressure is 30psi, the ionization voltage is 5500V, the temperature is 550 ℃, the spraying gas pressure is 9psi, the spraying gas pressure is 55psi, the auxiliary heating gas pressure is 55psi, and the measuring method is as follows: preparing a reference substance solution and a test substance solution respectively, injecting the reference substance solution and the test substance solution into a high performance liquid chromatography-mass spectrometer, recording an MRM (molecular sieve) map, and calculating according to an external standard method and peak areas, wherein the preparation method of the reference substance solution comprises the following steps: taking a proper amount of impurity compound reference substances of the formulas II-1 and II-2, precisely weighing, adding acetonitrile for dissolving and diluting to prepare solutions with about 17.5ng of each 1mL, wherein the preparation method of the test sample solution comprises the following steps: accurately weighing 500mg of a sample, wherein the sample is oxcarbazepine or a compound sample of formula III, placing into a 10mL measuring flask, adding acetonitrile for dissolving and diluting to scale, shaking uniformly for use,
，
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 gradient elution procedure was:
，
the mass spectrometric detection ion pairs of the compound of formula II are:
，
the valve switching time in the mass spectrum detection is as follows:
。
2. the content detection method according to claim 1, wherein the high performance liquid chromatography-mass spectrometry detection method specifically comprises:
(1) liquid chromatography conditions:
chromatographic column: infinitylab poroshell 120 EC-C18,3mm×100mm,1.9 μm
Mobile phase: phase A is 0.1% formic acid aqueous solution, phase B is 95% acetonitrile aqueous solution
Flow rate: 0.3mL/min
Column temperature: 40 DEG C
Sample injection amount: 1 mu L
Gradient elution conditions:
，
(2) mass spectrometry conditions:
ion source: electrospray ionization source
Monitoring mode: multiple reaction monitoring mode
Detection mode: positive ion mode
Air curtain air pressure: 30psi
Ionization voltage of 5500V
The temperature is 550 DEG C
The impinging air pressure was 9psi
The spray gas pressure was 55psi
The auxiliary heating air pressure is 55psi
The ion pairs are:
，
valve switching time:
，
(3) the measuring method comprises the following steps:
control solution: taking a proper amount of impurity compound reference substances of the formulas II-1 and II-2, precisely weighing, adding acetonitrile for dissolving and diluting to prepare solutions with about 17.5ng each 1 mL;
test solution: precisely weighing 500mg of a sample, wherein the sample is oxcarbazepine or a compound sample of formula III, placing the sample in a 10mL measuring flask, adding acetonitrile for dissolution, diluting to a scale, and shaking uniformly for later use;
precisely measuring 1 μl of each of the sample solution and the reference solution, respectively injecting into high performance liquid chromatography-mass spectrometry detector according to the detection conditions, recording MRM, and calculating with peak area according to external standard method.
3. Use of the content detection method according to any one of claims 1-2 for the high sensitivity detection of the content of oxcarbazepine key starting material compound of formula II.
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