CN118688370B - A quality control method for new antiviral drug intermediates - Google Patents
A quality control method for new antiviral drug intermediates Download PDFInfo
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- 238000003908 quality control method Methods 0.000 title claims abstract description 20
- 239000003443 antiviral agent Substances 0.000 title claims description 12
- 239000000543 intermediate Substances 0.000 title description 27
- 239000012535 impurity Substances 0.000 claims abstract description 46
- 230000000840 anti-viral effect Effects 0.000 claims abstract description 23
- 239000000126 substance Substances 0.000 claims abstract description 22
- 239000002547 new drug Substances 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- 239000003960 organic solvent Substances 0.000 claims abstract description 5
- 150000001875 compounds Chemical class 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 44
- 239000000243 solution Substances 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 26
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 14
- 239000003480 eluent Substances 0.000 claims description 11
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000004007 reversed phase HPLC Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 14
- 239000012467 final product Substances 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 37
- 239000000523 sample Substances 0.000 description 27
- 239000003085 diluting agent Substances 0.000 description 13
- 238000005303 weighing Methods 0.000 description 10
- 239000003814 drug Substances 0.000 description 7
- 239000012488 sample solution Substances 0.000 description 7
- 239000011259 mixed solution Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000013558 reference substance Substances 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 5
- 238000007865 diluting Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- -1 phenylmethyloxy Chemical group 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- FIQMHBFVRAXMOP-UHFFFAOYSA-N triphenylphosphane oxide Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)(=O)C1=CC=CC=C1 FIQMHBFVRAXMOP-UHFFFAOYSA-N 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 101000610640 Homo sapiens U4/U6 small nuclear ribonucleoprotein Prp3 Proteins 0.000 description 1
- 101001110823 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-A Proteins 0.000 description 1
- 101000712176 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-B Proteins 0.000 description 1
- 102100040374 U4/U6 small nuclear ribonucleoprotein Prp3 Human genes 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000037797 influenza A Diseases 0.000 description 1
- 208000037798 influenza B Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to the technical field of compound detection, in particular to a quality control method of an antiviral new drug intermediate, which comprises the following steps of dissolving an intermediate sample in an organic solvent to obtain an intermediate solution; and taking the intermediate solution, and detecting impurities by high performance liquid chromatography. The quality control method of the antiviral new drug intermediate provided by the invention can effectively separate the antiviral new drug intermediate from adjacent impurities and known impurities, accurately measure the impurity content, has high separation degree, and effectively solves the problem of difficult separation and detection of the antiviral new drug intermediate and related substances, thereby ensuring the quality controllability of the antiviral new drug intermediate and further providing powerful guarantee for the quality control of the antiviral new drug of the final product.
Description
Technical Field
The invention relates to the technical field of compound detection, in particular to a quality control method of an antiviral new drug intermediate.
Background
WXSH0208 is a class 1 inhibitor of small molecule RNA polymerase PA subunit, and is currently in clinical stage III, and the indication is the treatment of influenza A and B. The chemical structural formula of WXSH0208 antiviral new drug intermediate WXSH0208-3 is as follows:
Chemical name (7 aS, 16R) -10- (phenylmethyloxy) -2, 3-difluoro-8-methyl-16-phenyl-6, 7a, 8-tetrahydro-16H-benzo [ b ] pyrido [1',2':1,6] [1,2,4] triazinyl [2,3-e ] [1,5] oxazosin-9, 11-dione.
WXSH0208 the 0208-3 intermediate may include various related substances (including 16 impurities, see the summary of the impurities in the related substances in table 1 for details) as impurities derived from starting materials, intermediates, process impurities, byproducts, degradation impurities and the like, and in order to ensure the quality of the WXSH the 0208-3 intermediate, it is necessary to perform qualitative and quantitative analysis on the related substances.
TABLE 1 impurity summary table in antiviral New drug intermediates
Disclosure of Invention
The present invention is directed to solving at least one of the technical problems existing in the related art. Therefore, the invention aims to provide a quality control method of an antiviral new drug intermediate.
In order to achieve the purpose, the invention adopts the following technical scheme:
a quality control method of an antiviral new drug intermediate comprises the following steps:
;
the method comprises the following steps:
s100, taking the intermediate sample and dissolving the intermediate sample in an organic solvent to obtain the intermediate solution;
s200, taking the intermediate solution, and detecting impurities by high performance liquid chromatography;
the impurities include at least one of the following compounds:
、、、、、、、、、、、、、、 And 。
Further, the high performance liquid chromatography is reversed phase high performance liquid chromatography.
Further, the chromatographic conditions of the reversed phase high performance liquid chromatography are as follows:
the chromatographic column is C18 chromatographic column with specification of 4.6X105 mm and 3.5 μm;
the mobile phase is that 0.1% phosphoric acid aqueous solution is used as mobile phase A, methanol is used as mobile phase B, and gradient elution is adopted;
the detection wavelength is 220nm;
The flow rate is 0.7 ml/min-0.9 ml/min;
the column temperature is 38-42 ℃.
Further, the gradient elution process comprises the steps of keeping the volume ratio of the mobile phase A in an eluent at 70% and the volume ratio of the mobile phase B in 30% within 0 min-15 min, uniformly changing the eluent to 40min, keeping the volume ratio of the mobile phase A at 25% and the volume ratio of the mobile phase B at 75%, uniformly changing the eluent to 45min, keeping the volume ratio of the mobile phase A at 10% within 45 min-50 min, keeping the volume ratio of the mobile phase B at 90%, uniformly changing the eluent to 50.1min, keeping the volume ratio of the mobile phase A at 70% and keeping the volume ratio of the mobile phase B at 30% within 50.1 min-60 min.
Further, the gradient elution process comprises the steps of keeping the volume ratio of the mobile phase A of the eluent at 70% and the volume ratio of the mobile phase B at 30% within 0 min-5 min, uniformly changing the eluent to 25min, keeping the volume ratio of the mobile phase A at 50% and the volume ratio of the mobile phase B at 50%, uniformly changing the eluent to 55min, keeping the volume ratio of the mobile phase A at 40% and the volume ratio of the mobile phase B at 60%, uniformly changing the eluent to 70min, keeping the volume ratio of the mobile phase A at 10% and the volume ratio of the mobile phase B at 90%, uniformly changing the eluent to 70.1min, keeping the volume ratio of the mobile phase A at 70% and the volume ratio of the mobile phase B at 30% within 70.1 min-80 min.
Further, the gradient elution process comprises the steps of keeping the volume ratio of the mobile phase A at 70% and the volume ratio of the mobile phase B at 30% within 0 min-5 min, uniformly changing the time to 25min, keeping the volume ratio of the mobile phase A at 50% and the volume ratio of the mobile phase B at 50%, uniformly changing the time to 55min, keeping the volume ratio of the mobile phase A at 40% and the volume ratio of the mobile phase B at 60%, uniformly changing the time to 70min, keeping the volume ratio of the mobile phase A at 10% within 70 min-80 min, keeping the volume ratio of the mobile phase B at 90%, uniformly changing the time to 80.1min, keeping the volume ratio of the mobile phase B at 70% within 80.1 min-90 min, and keeping the volume ratio of the mobile phase B at 30%.
Further, the flow rate was 0.8ml/min.
Further, the column temperature was 40 ℃.
Further, in S100, the organic solvent is dichloromethane.
Further, a ghost peak trapping column is additionally arranged and used for eliminating the interference of the solvent and gradient change peaks.
The above technical solutions in the embodiments of the present invention have at least one of the following technical effects:
The quality control method of the antiviral new drug intermediate provided by the invention can effectively separate the antiviral new drug intermediate from adjacent impurities and known impurities, accurately measure the impurity content, has high separation degree, and effectively solves the problem of difficult separation and detection of the antiviral new drug intermediate and related substances thereof, thereby ensuring the quality controllability of the antiviral new drug intermediate.
The quality control method of the antiviral new drug intermediate provided by the invention has the advantages of high sensitivity and good accuracy, can more comprehensively carry out accurate quantitative detection on each impurity in the sample, and establishes reasonable control limits, thereby providing powerful guarantee for quality control of the antiviral new drug WXSH0208 of the final product.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chromatogram of a mixed solution of an intermediate of an antiviral agent and 5 related substances in example 1 of the present invention.
FIG. 2 is a chromatogram of a mixed solution of an intermediate of an antiviral agent and 5 related substances in example 2 of the present invention.
FIG. 3 is a chromatogram of a mixed solution of an intermediate of an antiviral agent and 16 related substances in example 3 of the present invention.
FIG. 4 is a chromatogram of a mixed solution of an antiviral novel drug intermediate and 14 related substances in example 4 of the present invention.
FIG. 5 is a chromatogram of a mixed solution of an intermediate of an antiviral agent and 16 related substances in example 5 of the present invention.
FIG. 6 is a chromatogram for detecting impurity of related substances in example 6 of the present invention.
FIG. 7 is a quantitative limit chromatogram of impurities of a related substance in example 6 of the present invention.
FIG. 8 is a chromatogram of a 100% horizontal loading solution in example 6 of the present invention.
Wherein WXSH0208-3 represents an antiviral novel drug intermediate.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The following examples are illustrative of the invention but are not intended to limit the scope of the invention.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
In the following examples and comparative examples, the drugs, reagents and instruments used were as follows:
High performance liquid chromatograph (Vanquish-DAD, thermoFisher), electronic balance (XPR 205, metrele Toli Instrument Shanghai Co., ltd.), electronic balance (MSX (SG EA), sidolis Co.);
XBIdge C18, column (4.6X105 mm,3.5 μm) (Waters), waters XBridge Shield RP Column (4.6X105 mm,3.5 μm) (Waters), ghost-Sniper Column (Ghost-Column (4.0X130 mm)), and Mr. chromatography;
Reagents, methanol (chromatographic purity, lot number 240102, conscode), acetonitrile (chromatographic purity, lot number 240109, conscode), phosphoric acid (chromatographic purity, lot number 20240110, komi Europe), water (ultrapure water, homemade);
WXSH0208-BP33a (Z-1) (lot number: E040641-030-04, source: du Ji Qing Zhi Ji Zhi Co., ltd.);
WXSH0208-BP104 (Z-2) (lot number: E040639-019-09, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-BP27 (Z-3) (lot number: WS-ES18228-492-P1, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-BP12 (Z-4) (lot number: E040639-048-01, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-4 (Z-5) (lot number: WS-QC00003270-07-K-202312001, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-SM1 (Z-6) (lot number: WS-WXSH0208-SM1-240119-1, source: tianjin Chenxin medicine research Co., ltd.);
TPPO (Z-7) (lot 972DRF4R, source An Naiji chemical);
WXSH0208-BP108b (Z-8) (lot number: E040639-052-04, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-BP30 (Z-9) (lot number: WS-WXSH0208-BP30-240511-1, source: tianjin chenxin medicine research Co., ltd.);
WXSH0208-208-107a (Z-10) (lot number: E040638-093-01, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-BP106b (Z-11) (lot number: E040639-056-01, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-1 (Z-12) (lot number: WS-WXSH0208-1-52220737011-1AA, source: tianjin chenxin medicine research Co., ltd.);
WXSH0208-1A (Z-13) (lot number: WS-QC00003270-05-K-202311002, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-BP5 (Z-14) (lot number: E040638-104-01, source: du Ji Zhi Ji Co., ltd.);
WXSH0208-BP24a (Z-15) (lot number: ES18228-494-P1, source: shanghai Minkangde New drug development Co., ltd.);
WXSH0208-BP24b (Z-16) (lot number: ES18228-494-P2, source: shanghai Minkangde New drug development Co., ltd.);
An antiviral intermediate (WXSH 0208-3) sample (batch number: E040583-132-01, source: duchuang (Chongqing) pharmaceutical technology Co., ltd.).
Example 1
The instrument and conditions are as follows:
the chromatographic column is RP18 chromatographic column with the specification of 4.6X105 mm and 3.5 μm;
mobile phase A is 0.1% phosphoric acid aqueous solution and B is methanol;
the gradient elution process is as follows:
the flow rate is 0.8ml/min;
Column temperature is 40 ℃;
The detection wavelength is 220nm;
The sample loading was 6. Mu.l.
Diluent water-methanol (volume ratio 20:80)
The preparation method comprises the steps of weighing WXSH0208-3 samples about 20mg in a 10ml measuring flask, adding 1ml of dichloromethane (Dichloromethane, DCM for short) to dissolve, adding 2ml of methanol, respectively adding impurities Z-2, Z-4, Z-5, Z-7 and Z-11 according to 0.2% of the concentration of the sample, diluting to scale by adding a diluent, and uniformly mixing.
As shown in FIG. 1, the method is used for detecting WXSH0208-3 related substances, Z-7 and Z-2 do not reach baseline separation, the separation degree is 1.84, Z-11 is not separated from the previous impurities, and the separation degree is 1.31, so that the chromatographic column is tried to be replaced.
Example 2
The instrument and conditions are as follows:
the chromatographic column is C18 chromatographic column with specification of 4.6X105 mm and 3.5 μm;
mobile phase A is 0.1% phosphoric acid aqueous solution and B is methanol;
the gradient elution process is as follows:
the flow rate is 0.8ml/min;
Column temperature is 40 ℃;
The detection wavelength is 220nm;
The sample loading was 6. Mu.l.
Diluent water-methanol (volume ratio 20:80)
The operation steps are that about 20mg of WXSH0208-3 sample is weighed in a 10ml measuring flask, 1ml of dichloromethane is added to dissolve, 2ml of methanol is added, the impurities Z-2, Z-4, Z-5, Z-7 and Z-11 are respectively added according to 0.2 percent of the concentration of the sample, and then the mixture is diluted to scale by adding a diluent, and the mixture is uniformly mixed, thus obtaining the product.
As shown in figure 2, the method is used for detecting WXSH0208-3 related substances, and the separation effect of each impurity is good.
Example 3
The instrument and conditions are as follows:
the chromatographic column is C18 chromatographic column with specification of 4.6X105 mm and 3.5 μm;
mobile phase A is 0.1% phosphoric acid aqueous solution and B is methanol;
the gradient elution process is as follows:
the flow rate is 0.8ml/min;
Column temperature is 40 ℃;
The detection wavelength is 220nm;
The sample loading was 6. Mu.l.
Diluent water-methanol (volume ratio 20:80)
The operation steps are that about 20mg of the sample to be tested is weighed and put into a 10ml measuring flask, 1ml of dichloromethane is added to dissolve, 2ml of methanol is added to dilute, and the impurities Z-1, Z-2, Z-3, Z-4, Z-5, Z-6, Z-7, Z-8, Z-9, Z-10, Z-11, Z-12, Z-13, Z-14, Z-15 and Z-16 are respectively added, and then 80% methanol is added to dilute and fix the volume to scale, and shaking is carried out, thus obtaining the product.
Wherein, each impurity is added according to 0.2% of the concentration of the sample.
And the detection result is that the chromatogram of the mixed solution is shown as figure 3, and the method is used for detecting WXSH0208-3 related substances, and the main peak is not separated from the impurities before and after the main peak, so that the gradient is continuously optimized.
Example 4
The instrument and conditions are as follows:
the chromatographic column is C18 chromatographic column with specification of 4.6X105 mm and 3.5 μm;
mobile phase A is 0.1% phosphoric acid aqueous solution and B is methanol;
the gradient elution process is as follows:
the flow rate is 0.8ml/min;
Column temperature is 40 ℃;
The detection wavelength is 220nm;
The sample loading was 6. Mu.l.
Diluent water-methanol (volume ratio 20:80)
The operation steps are that about 20mg of the sample to be tested is weighed and put into a 10ml measuring flask, 1ml of dichloromethane is added to dissolve, 2ml of methanol is added to dilute, and the impurities Z-1, Z-2, Z-3, Z-4, Z-5, Z-7, Z-9, Z-10, Z-11, Z-12, Z-13, Z-14, Z-15 and Z-16 are respectively added, and then 80% methanol is added to dilute and volume to scale, and shaking is carried out, thus obtaining the product.
Wherein, each impurity is added according to 0.2% of the concentration of the sample.
As shown in figure 4, the method is used for detecting WXSH0208-3 related substances, and has good impurity separation effect, so that small polar impurities in a sample can be eluted, and the elution time of 90% of methanol can be prolonged.
Example 5
Instrument and conditions.
The chromatographic column is C18 chromatographic column with specification of 4.6X105 mm and 3.5 μm;
mobile phase A is 0.1% phosphoric acid aqueous solution and B is methanol;
the gradient elution process is as follows:
the flow rate is 0.8ml/min;
Column temperature is 40 ℃;
The detection wavelength is 220nm;
the sample loading was 10. Mu.l.
Diluent water-methanol (volume ratio 20:80)
The operation steps are as follows:
Weighing about 20mg of the sample solution, placing in a 10ml measuring flask, adding 1ml of dichloromethane to dissolve, adding 2ml of methanol for dilution, adding 80% methanol for dilution and volume fixing to scale, and shaking uniformly to obtain the final product.
And (3) mixing the solution, namely weighing about 20mg of the sample to be tested, placing the sample into a 10ml measuring flask, adding 1ml of dichloromethane to dissolve the sample, adding 2ml of methanol to dilute the solution, respectively adding proper amounts of impurities Z-1, Z-2, Z-3, Z-4, Z-5, Z-6, Z-7, Z-8, Z-9, Z-10, Z-11, Z-12, Z-13, Z-14, Z-15 and Z-16, adding 80% of methanol to dilute the solution to a certain volume to scale, and shaking the solution uniformly to obtain the finished product.
Wherein, Z-9 is added according to 0.1 percent, and the other impurities are added according to 0.2 percent of the concentration of the test sample.
As shown in FIG. 5, the method is used for detecting WXSH0208-3 related substances, impurities are eluted at 77.342min, and the separation degree of the impurities is good.
And (II) examining the durability range of the method parameters.
The durability range of the method is examined from the conditions of the initial proportion of the mobile phase organic phase, the column temperature, the flow rate and the like, and the durability range is shown in the following table:
The column temperature is 38-42 ℃ and the initial proportion of the organic phase is 28-32%.
The column temperatures of 38 ℃, 40 ℃ and 42 ℃ are sequentially examined, the initial proportion of the organic phase is 28%, 30% and 32%, the higher the column temperature is, the higher the initial proportion of the organic phase is, the peak time is advanced, the separation degree between impurities is reduced, and the separation can meet the detection requirement.
The flow rate range is 0.7ml/min to 0.9ml/min.
Under the same conditions, the higher the flow rate is, the earlier the peak time is, and under the conditions of 3 flow rates, the separation between each impurity and the sample can meet the requirements, preferably the separation is better, and the flow rate of 0.8ml/min with proper peak time is obtained.
Example 6
And (one) examining the quantitative limit and the detection limit.
Chromatographic conditions the same chromatographic conditions as used in example 5 were used.
The operation steps comprise precisely weighing the right amount of each impurity reference substance, precisely weighing, adding methanol for dissolving and diluting to scale to obtain each impurity reference substance solution, gradually diluting to prepare each impurity quantitative limit and detection limit solution, injecting into a liquid chromatograph, recording chromatogram, and detecting chromatogram as shown in figure 6 and figure 7.
The quantitative limit and the detection limit of each impurity are shown in the following table:
The method is used for detecting WXSH0208-3 related substances, the detection limit is less than 0.010 percent according to the concentration of the solution of the test sample being 2mg/ml, the sensitivity is higher, and the detection requirements are met.
And (II) examining the recovery rate.
Chromatographic conditions the optimal process conditions were determined as in example 5.
The procedure was carried out by examining the recovery rates at 50%, 100% and 150% (the amounts of each impurity having about 50%, 100% and 150% limits were added to the test sample solutions, respectively, and the sample addition recovery rates for each impurity were calculated).
The reference substance solution is prepared by precisely weighing a proper amount of each impurity reference substance, precisely weighing, adding diluent for dissolving and diluting to obtain a solution containing about WXSH0208-BP30 (Z-9) 10 mug and 20 mug of the rest impurities per ml, and the solution is used as a mixed reference substance stock solution. Then precisely measuring 2ml of mixed reference stock solution, placing the mixed reference stock solution in a 10ml measuring flask, adding 1ml of dichloromethane, and then adding a diluent to dilute to scale to obtain a reference solution.
Accurately weighing WXSH0208-3 samples about 20mg, placing the samples in a 10ml measuring flask, adding 1ml of dichloromethane to dissolve the samples, adding 2ml of methanol to mix the samples uniformly, and adding a diluent to scale to obtain a sample solution.
About 20mg of WXSH0208-3 samples are precisely weighed, placed in a 10ml measuring flask, 1ml of dichloromethane is added to dissolve, 2ml of methanol is added to mix evenly, 1ml of mixed reference stock solution is precisely measured in the flask, and the diluent is added to scale to serve as 50% horizontal sample adding solution.
Accurately weighing WXSH0208-3 samples about 20mg, placing the samples in a 10ml measuring flask, adding 1ml of dichloromethane to dissolve the samples, adding 2ml of methanol to mix the samples uniformly, accurately weighing 2ml of mixed reference stock solution in the flask, and adding a diluent to scale to obtain 100% horizontal sample solution.
150% Level sample solution about 20mg of WXSH0208-3 sample is precisely weighed, placed in a 10ml measuring flask, 1ml of dichloromethane is added to dissolve, 2ml of methanol is added to mix evenly, 3ml of mixed reference stock solution is precisely measured in the flask, and diluent is added to scale to serve as 150% level sample solution.
And respectively measuring a reference substance solution, a sample solution and a sample adding solution, injecting into a liquid chromatograph, recording a chromatogram, and calculating the recovery rate.
The 100% level loading solution detection profile is shown in figure 8.
The results of recovery at each level are shown in the following table:
as shown in the table, 16 impurities are in the range of 50% -150%, the average recovery rate is 85% -110%, the requirements are met, and the quality control method of the antiviral novel drug intermediate is proved to be good in accuracy.
It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.
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| CN115144509A (en) * | 2022-09-05 | 2022-10-04 | 天津辰欣药物研究有限公司 | Quality control method of diquafoso tetrasodium intermediate 5' -uridylate disodium |
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