CN111665297B - Separation detection method of Baloxavir marboxil optical isomer - Google Patents
Separation detection method of Baloxavir marboxil optical isomer Download PDFInfo
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- CN111665297B CN111665297B CN201910162182.5A CN201910162182A CN111665297B CN 111665297 B CN111665297 B CN 111665297B CN 201910162182 A CN201910162182 A CN 201910162182A CN 111665297 B CN111665297 B CN 111665297B
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- RZVPBGBYGMDSBG-GGAORHGYSA-N baloxavir marboxil Chemical compound COC(=O)OCOc1c2C(=O)N3CCOC[C@H]3N([C@H]3c4ccc(F)c(F)c4CSc4ccccc34)n2ccc1=O RZVPBGBYGMDSBG-GGAORHGYSA-N 0.000 title claims abstract description 95
- 229940008411 baloxavir marboxil Drugs 0.000 title claims abstract description 88
- 238000001514 detection method Methods 0.000 title claims abstract description 35
- 238000000926 separation method Methods 0.000 title claims abstract description 28
- 230000003287 optical effect Effects 0.000 title claims abstract description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 90
- 150000001335 aliphatic alkanes Chemical class 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000000945 filler Substances 0.000 claims abstract description 6
- 150000004676 glycans Chemical class 0.000 claims abstract description 6
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 6
- 239000005017 polysaccharide Substances 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 5
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 64
- 239000000243 solution Substances 0.000 claims description 43
- 239000003085 diluting agent Substances 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 22
- 101000652482 Homo sapiens TBC1 domain family member 8 Proteins 0.000 claims description 7
- 102100030302 TBC1 domain family member 8 Human genes 0.000 claims description 7
- 239000012046 mixed solvent Substances 0.000 claims description 6
- 150000007524 organic acids Chemical class 0.000 claims description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 34
- 239000000523 sample Substances 0.000 description 34
- 230000014759 maintenance of location Effects 0.000 description 32
- FIDLLEYNNRGVFR-CTNGQTDRSA-N (3R)-2-[(11S)-7,8-difluoro-6,11-dihydrobenzo[c][1]benzothiepin-11-yl]-11-hydroxy-5-oxa-1,2,8-triazatricyclo[8.4.0.03,8]tetradeca-10,13-diene-9,12-dione Chemical compound OC1=C2N(C=CC1=O)N([C@@H]1COCCN1C2=O)[C@@H]1C2=C(SCC3=C1C=CC(F)=C3F)C=CC=C2 FIDLLEYNNRGVFR-CTNGQTDRSA-N 0.000 description 17
- 150000001875 compounds Chemical class 0.000 description 14
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 238000010828 elution Methods 0.000 description 11
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 239000012490 blank solution Substances 0.000 description 9
- 238000003821 enantio-separation Methods 0.000 description 9
- 239000012535 impurity Substances 0.000 description 9
- 238000012544 monitoring process Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 239000012488 sample solution Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 4
- CXHHBNMLPJOKQD-UHFFFAOYSA-M methyl carbonate Chemical compound COC([O-])=O CXHHBNMLPJOKQD-UHFFFAOYSA-M 0.000 description 4
- KPCOLEDDUNYSQA-UHFFFAOYSA-N (3,5-dimethylphenyl)carbamic acid Chemical compound CC1=CC(C)=CC(NC(O)=O)=C1 KPCOLEDDUNYSQA-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- KBRZBBOTZJFKFH-UHFFFAOYSA-N (3,5-dichlorophenyl) carbamate Chemical compound NC(=O)OC1=CC(Cl)=CC(Cl)=C1 KBRZBBOTZJFKFH-UHFFFAOYSA-N 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 229940123734 Endonuclease inhibitor Drugs 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 208000037797 influenza A Diseases 0.000 description 1
- 208000037798 influenza B Diseases 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- LPNBBFKOUUSUDB-UHFFFAOYSA-M p-toluate Chemical compound CC1=CC=C(C([O-])=O)C=C1 LPNBBFKOUUSUDB-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- -1 trifluoroacetic acid Chemical class 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to a separation and detection method of optical isomers, and belongs to the field of analytical chemistry. The method specifically relates to a chiral chromatographic column using polysaccharide derivatives as a filler and a mixed solution of lower alkane and lower alcohol as a mobile phase, wherein the chiral chromatographic column is used for separating and measuring Baloxavir marboxil and optical isomers thereof by using a liquid chromatography. The method is simple, quick and accurate.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for separating and measuring Baloxavir marboxil isomers by liquid chromatography.
Background
XOFLUZA (baloxavir marboxil) is an antiviral PA endonuclease inhibitor which inhibits initiation of mRNA synthesis and can be used for treating influenza A and B in adults and children. Baloxavir marboxil methyl ({ (12 aR) -12- [ (11S) -7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiazepin 11-yl ] -6, 8-dioxo-3, 4,6,8,12 a-hexahydro-1H- [1,4] oxodiazepino [3,4-C ] pyrido [2,1-f ] [1,2,4] triazin-7-yl } oxy) methyl carbonate, abbreviated herein as RS, having a molecular weight of 571.55,Baloxavir marboxil and a molecular formula of C 27H23F2N3O7 S, the chemical structure being as follows:
Baloxavir marboxil molecules have 2 chiral centers, and 4 optical isomers with different stereo configurations are generated simultaneously in chemical synthesis, and are listed as Baloxavir marboxil impurities, and the chemical names of the isomers are respectively as follows: ({ (12 aS) -12- [ (11R) -7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiazepin 11-yl ] -6, 8-dioxo-3, 4,6,8,12 a-hexahydro-1H- [1,4] oxodiazepin-yl [3,4-c ] pyrido [2,1-f ] [1,2,4] triazin-7-yl } oxy) methyl carbonate, abbreviated aS SR; ({ (12 aS) -12- [ (11S) -7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiazepin 11-yl ] -6, 8-dioxo-3, 4,6,8,12 a-hexahydro-1H- [1,4] oxodiazepin-yl [3,4-c ] pyrido [2,1-f ] [1,2,4] triazin-7-yl } oxy) methyl carbonate, abbreviated aS SS; ({ (12 aR) -12- [ (11R) -7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiazepin 11-yl ] -6, 8-dioxo-3, 4,6,8,12 a-hexahydro-1H- [1,4] oxodiazepin-yl [3,4-c ] pyrido [2,1-f ] [1,2,4] triazin-7-yl } oxy) methyl carbonate abbreviated as RR.
Quality control is required in the production Baloxavir marboxil process, but currently, no collection Baloxavir marboxil exists in the U.S. pharmacopoeia (USP), european Pharmacopoeia (EP), chinese pharmacopoeia (Ch.P.), and no detection method for separating and detecting the Baloxavir marboxil related literature report and the optical isomer thereof is found. In order to better and more accurately control the content of optical isomer in the product and ensure the quality of the bulk drug and the preparation product, the invention provides an analysis method suitable for Baloxavir marboxil and the content measurement of the optical isomer thereof. The method can simply, rapidly and accurately separate and detect Baloxavir marboxil and the optical isomer thereof.
Disclosure of Invention
The invention aims to provide a method for separating and measuring Baloxavir marboxil and optical isomer thereof by using a chiral chromatographic column, thereby realizing the separation and measurement of Baloxavir marboxil and optical isomer thereof.
A method for separating and measuring Baloxavir marboxil and optical isomer thereof by liquid chromatography is characterized in that chiral chromatographic columns with polysaccharide derivatives as fillers are adopted, and mixed solution of lower alkane and lower alcohol is adopted as mobile phase.
In some embodiments, the polysaccharide derivative filler of the chiral chromatography column is amylose-tris (3, 5-xylylcarbamate), amylose-tris [ (S) - α -tolylcarbamate ], cellulose-tris (3, 5-xylylcarbamate), cellulose-tris [ 4-methylbenzoate ], or cellulose-tris (3, 5-dichlorophenyl carbamate), or a combination thereof. In certain embodiments, the polysaccharide derivative filler is amylose-tris (3, 5-xylylcarbamate).
The chiral chromatography column may be selected from CHIRALPAK AD-3, CHIRALPAK AD, CHIRALPAK AS-H, CHIRALCELOD-H, CHIRALCEL OJ-H, or CHIRALPAK IC. In some embodiments, the chiral chromatography column is CHIRALPAK AD, particularly CHIRALPAK AD-H or CHIRALPAK AD-3, and the vendor is Daxiliao chiral technology (Shanghai) Inc., english name DAICEL CHIRAL TECHNOLOGIES (CHINA) CO., LTD. In some embodiments, the chiral chromatography column is CHIRALPAK AD-3.
In some embodiments, the lower alkane is a common alkane, including n-hexane, n-pentane, n-heptane, or cyclohexane, or a combination thereof, in certain embodiments n-hexane.
In some embodiments, the mobile phase may further contain an organic acid. In some embodiments, the mobile phase contains an organic acid, such as trifluoroacetic acid (TFA), or the like. In some embodiments, the mobile phase lower alcohol solution may contain an organic acid (e.g., trifluoroacetic acid, etc.) in a volume percent of about 0.05% to about 0.5% to lower alcohol.
In some embodiments, the lower alcohol is methanol, ethanol, propanol, n-butanol, isopropanol, or a combination thereof. In certain embodiments the lower alcohol is ethanol.
In some embodiments, the volume ratio of mobile phase lower alkane to lower alcohol solution is from 60:40 to 80:20. In some embodiments, the volume ratio of mobile phase lower alkane to lower alcohol solution is 70:30. In some embodiments, the volume ratio of mobile phase lower alkane to lower alcohol solution is 60:40. In some embodiments, the mobile phase lower alkane to lower alcohol solution volume ratio is 80:20.
In some embodiments, the separation assay of the present invention can be implemented as follows:
1) Taking Baloxavir marboxil or a proper amount of a sample containing Baloxavir marboxil, and dissolving the sample by using a certain amount of mixed solution of lower alkane and lower alcohol as a diluent;
2) Setting instrument parameters: flow rate of mobile phase, detection wavelength, column box temperature of chromatographic column;
3) Taking a certain amount of the solution in the step 1), and injecting the solution into a high performance liquid chromatograph to finish Baloxavir marboxil and the separation and determination of the optical isomer thereof.
Baloxavir marboxil as described in step 1) may be of any purity.
Baloxavir marboxil as described in step 1) may be of any optical purity.
The lower alkane of the diluent in the step 1) is n-hexane, n-pentane, n-heptane or cyclohexane or a combination thereof, and the lower alcohol solvent is methanol, ethanol, n-propanol, n-butanol, isopropanol or a combination thereof. In some embodiments, in the diluent, the lower alkane is n-hexane; in some embodiments, the lower alcohol solvent is ethanol. In certain embodiments, the mixed solvent of lower alkane and lower alcohol is a mixed solvent of n-hexane and ethanol; the volume ratio (V/V) of the lower alkane to the lower alcohol is from 50:50 to 10:90. In some embodiments, the volume ratio (V/V) of the lower alkane to lower alcohol in the diluent is 1:4.
Each 1ml of the diluent contains Baloxavir marboxil mg-4 mg of sample. In some embodiments, the diluent of step 1) contains Baloxavir marboxil samples of 2mg per 1ml of diluent.
The flow rate of the mobile phase is 0.5 ml/min-1.5 ml/min. In some embodiments, the flow rate of the mobile phase is 0.5ml/min; in some embodiments, the flow rate of the mobile phase is 0.8ml/min; in some embodiments, the flow rate of the mobile phase is 1.0ml/min.
The detection wavelength is 200nm to 230nm. In some embodiments, the detection wavelength is 210nm.
The temperature of the chromatographic column box is 25-40 ℃. In some embodiments, the column box temperature is 25 ℃; in some embodiments, the column box temperature is 35 ℃; in some embodiments, the column box temperature is 38 ℃.
The sample injection amount of the sample solution is 2-20 mu l. In some embodiments, the sample solution is introduced at 5 μl. In some embodiments, the sample solution is introduced in an amount of 10 μl.
In some embodiments, the separation assay methods of the present invention can be implemented as follows:
1) Taking Baloxavir marboxil or a proper amount of a sample containing Baloxavir marboxil, dissolving the sample by using a certain amount of a mixed solvent of lower alkane and lower alcohol as a diluent, and preparing a sample solution containing Baloxavir marboxil 0.2.2-4 mg per 1 ml;
2) Setting the flow rate of the mobile phase to be 0.5 ml/min-1.5 ml/min, the detection wavelength to be 200 nm-230 nm, and the temperature of a chromatographic column box to be 25-40 ℃;
3) Taking 2-20 mu l of the sample solution in the step 1), and injecting into a high performance liquid chromatograph to finish Baloxavir marboxil and the separation and measurement of the optical isomer thereof.
In the method provided by the invention, the high performance liquid chromatograph can be an Agilent 1260 type high performance liquid chromatograph system and a workstation in the United states.
In some embodiments, the chiral chromatography column is CHIRALPAK AD-3, the mobile phase is n-hexane to ethanol (0.3% TFA, V/V), and the volume ratio (V/V) is about 70:30.
In some embodiments, the chiral chromatography column is CHIRALPAK AD-3, the mobile phase is n-hexane to ethanol (0.3% TFA, V/V), and the volume ratio (V/V) is about 80:20.
In some embodiments, the chiral chromatography column is CHIRALPAK AD-3, the mobile phase is n-hexane to ethanol (0.3% TFA, V/V), and the volume ratio (V/V) is about 60:40.
In some embodiments, the chiral chromatography column is CHIRALPAK AD-H and the mobile phase is n-hexane to ethanol (0.3% TFA, V/V) at a volume ratio (V/V) of about 70:30.
In some embodiments, the chiral chromatography column is CHIRALPAK AD, the mobile phase is n-hexane to ethanol (0.3% TFA, V/V), and the volume ratio (V/V) is about 70:30.
With the separation method of the present invention, the time for separating the assay Baloxavir marboxil and its optical isomers is within 50 minutes, in some embodiments within 40 minutes, and in some embodiments within 30 minutes.
In the context of the foregoing or following, all numbers disclosed herein are approximations, whether or not the word "about" or "about" is used by the word "about". The numerical value of each number may vary by 1%, 2%, 5%, 7%, 8%, or 10%.
The invention adopts chiral chromatographic column with polysaccharide derivative as filler and mixed solution of lower alkane and lower alcohol as mobile phase, can effectively separate Baloxavir marboxil from enantiomer thereof, and has a separation degree of more than 1.5 or more than 2.5 or more than 5, and can completely separate the base line, thereby accurately and effectively controlling the quality of Baloxavir marboxil. The method can simply, rapidly and accurately separate and detect Baloxavir marboxil and the optical isomer thereof.
Drawings
FIG. 1Baloxavir marboxil high performance liquid chromatography of enantiomer (SR configuration);
FIG. 2Baloxavir marboxil high performance liquid chromatography of enantiomer (SR configuration) and diastereomer (SS configuration);
FIG. 3Baloxavir marboxil high performance liquid chromatogram of the main compound (RS configuration) and diastereoisomers (RR configuration);
FIG. 4Baloxavir marboxil high performance liquid chromatogram of the main compound (RS configuration);
FIG. 5 shows a high performance liquid chromatogram of the separation detection of example 2;
FIG. 6 shows a high performance liquid chromatogram of the separation detection of example 3;
FIG. 7 shows a high performance liquid chromatogram of the separation detection of example 3;
FIG. 8 shows a high performance liquid chromatogram of the separation detection of example 4;
FIG. 9 shows a high performance liquid chromatogram of the separation detection of example 4;
FIG. 10 shows a high performance liquid chromatogram of the separation detection of example 5;
FIG. 11 shows a high performance liquid chromatogram of the separation detection of example 5;
FIG. 12 shows a high performance liquid chromatogram of the separation detection of example 6;
FIG. 13 shows a high performance liquid chromatogram of the separation detection of example 6;
FIG. 14 shows a high performance liquid chromatogram of the separation detection of example 7;
FIG. 15 shows a high performance liquid chromatogram of the separation detection of example 7;
FIG. 16 shows a high performance liquid chromatogram of the separation detection of example 8;
FIG. 17 shows a high performance liquid chromatogram of the separation detection of example 8;
FIG. 18 shows a high performance liquid chromatogram of the separation detection of example 9;
FIG. 19 shows a high performance liquid chromatogram of the separation detection of example 9;
in the figure, the abscissa indicates retention time, minutes (min); the ordinate represents the electrical signal, mAU.
Detailed Description
The embodiment of the invention discloses a method for separating and detecting Baloxavir marboxil and enantiomers thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the method described herein without departing from the spirit and scope of the invention.
The present invention will be described in detail with reference to examples.
Specification of instrument and chromatographic column: an Agilent 1260 type high performance liquid chromatography system and workstation; automatic sample injection;
Examples 1 to 9: baloxavir marboxil and enantiomer thereof separated and detected by CHIRALPAK AD chiral chromatographic column
Example 1
Instrument and conditions
Chromatographic column: CHIRALPAK AD-3.150 x 4.6mm,3 μm;
A detector: DAD (ultraviolet detector), detection wavelength 210nm;
Flow rate: 0.8mL/min;
Column temperature: 35 ℃;
sample injection amount: 5. Mu.L;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=70:30 (V: V)
Run time: for 40min;
diluent/blank solution: ethanol: n-hexane=4:1 (V: V);
experimental procedure
About 20mg of Baloxavir marboxil-configuration RS+RR sample is taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale with a diluent, and shaken well to obtain the configuration RS+RR solution.
About 20mg of Baloxavir marboxil-configuration SR+SS sample is taken, precisely weighed into a 10ml brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale, and shaken uniformly to obtain a configuration SR+SS solution.
About 20mg of Baloxavir marboxil enantiomer (configuration SR) sample was taken, precisely weighed into a 10ml brown volumetric flask, sonicated with diluent and diluted to scale, and shaken well as enantiomer localization solution.
About 20mg of a Baloxavir marboxil-configuration RS (main compound) sample is taken, precisely weighed into a10 ml brown volumetric flask, ultrasonically dissolved by a diluent, diluted to a scale, and shaken uniformly to serve as a test solution.
And respectively taking a configuration enantiomer positioning solution, a configuration SR+SS solution, an RS+RR solution and a test sample solution, performing high performance liquid chromatography analysis according to the conditions, and recording chromatograms, wherein the results are shown in figures 1, 2, 3 and 4. The chromatographic peak with a retention time of about 13.49 minutes in fig. 1 is that of Baloxavir marboxil enantiomer (SR configuration); the chromatographic peak with a retention time of about 17.86 minutes in fig. 2 is that of Baloxavir marboxil diastereomer SS configuration; the chromatographic peak with a retention time of about 20.55 minutes in FIG. 3 is for Baloxavir marboxil main compound (RS configuration) and the chromatographic peak with a retention time of about 27 minutes is for Baloxavir marboxil diastereomer RR configuration.
FIGS. 1-4 above demonstrate that this detection method is capable of separating the 4 configurational isomers of Baloxavir marboxil. The method can be used for Baloxavir marboxil and isomer separation determination thereof, thereby being used for quality monitoring.
Example 2
Instrument and conditions
Chromatographic column: CHIRALPAK AD-3.150 x 4.6mm,3 μm;
A detector: DAD (ultraviolet detector), detection wavelength 210nm;
flow rate: 1.0mL/min;
Column temperature: 35 ℃;
sample injection amount: 5. Mu.L;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=70:30 (V: V)
Run time: for 40min;
diluent/blank solution: ethanol: n-hexane=4:1 (V: V);
experimental procedure
About 20mg of Baloxavir marboxil mixed standard samples (containing Baloxavir marboxil and 3 optical isomers thereof) are respectively taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale by a diluent, and shaken uniformly to serve as mixed standard samples.
And carrying out high performance liquid chromatography analysis on Baloxavir marboxil mixed standard sample solutions according to the conditions, and recording a chromatogram, wherein the result is shown in figure 5.
The chromatographic peak with a retention time of about 11 minutes in FIG. 5 was the chromatographic peak for Baloxavir marboxil enantiomer (SR configuration), the chromatographic peak with a retention time of about 14 minutes was the chromatographic peak for Baloxavir marboxil diastereomer SS configuration, the chromatographic peak with a retention time of about 16 minutes was the main compound Baloxavir marboxil (RS configuration), and the chromatographic peak with a retention time of about 22 minutes was Baloxavir marboxil diastereomer RR configuration.
FIG. 5 demonstrates that Baloxavir marboxil can be separated from its 3 configurational impurities in a process with a flow rate of 1.0mL/min, a condition that can be used for isomer quality monitoring of Baloxavir marboxil.
Example 3
Instrument and chromatographic conditions:
Chromatographic column: CHIRALPAK AD-3.150 x 4.6mm,3 μm
A detector: DAD (ultraviolet detector)
Detection wavelength: 210nm;
flow rate: 0.8ml/min;
column temperature: 25 ℃;
sample injection amount: 5 μl;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=70:30 (V: V)
Run time: 60min;
Diluent/blank solution: ethanol: n-hexane=4:1 (V: V)
Experimental procedure
About 20mg of Baloxavir marboxil-configuration RS+RR sample is taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale with a diluent, and shaken well to obtain the configuration RS+RR solution.
About 20mg of Baloxavir marboxil-configuration SR+SS sample is taken, precisely weighed into a 10ml brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale, and shaken uniformly to obtain a configuration SR+SS solution.
Taking the configuration RS+RR solution and the configuration SR+SS solution, performing high performance liquid chromatography under the above conditions, and recording chromatograms, wherein the results are shown in FIG. 6 and FIG. 7.
The chromatographic peak with a retention time of about 25 minutes in fig. 6 is the chromatographic peak with the Baloxavir marboxil RS configuration (main compound); the chromatographic peak with a retention time of about 33 minutes is the one with Baloxavir marboxil RR configuration.
The chromatographic peak with a retention time of about 15 minutes in fig. 7 is the chromatographic peak with Baloxavir marboxil SR configuration; the chromatographic peak with retention time of about 20 minutes is the one with Baloxavir marboxil SS configuration.
Figures 6 and 7 demonstrate that the Baloxavir marboxil main peak and the other 3 configurational impurities can be separated at column temperature of 25 c by 4 chromatographic peaks, a condition which can be used for isomer monitoring of Baloxavir marboxil.
Example 4
Instrument and chromatographic conditions:
Chromatographic column: CHIRALPAK AD-3.150 x 4.6mm,3 μm
A detector: DAD (ultraviolet detector)
Detection wavelength: 210nm;
flow rate: 0.8ml/min;
Column temperature: 35 ℃;
sample injection amount: 5 μl;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=80:20 (V: V)
Run time: for 40min;
diluent/blank solution: ethanol: n-hexane=4:1 (V: V);
experimental procedure
About 20mg of Baloxavir marboxil-configuration RS+RR sample is taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale with a diluent, and shaken well to obtain the configuration RS+RR solution.
About 20mg of Baloxavir marboxil-configuration SR+SS sample is taken, precisely weighed into a 10ml brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale, and shaken uniformly to obtain a configuration SR+SS solution.
Taking the configuration RS+RR solution and the configuration SR+SS solution, performing high performance liquid chromatography under the above conditions, and recording chromatograms, wherein the results are shown in FIG. 8 and FIG. 9.
The chromatographic peak with a retention time of about 28 minutes in fig. 8 is the chromatographic peak with the Baloxavir marboxil RS configuration (main compound); the chromatographic peak with a retention time of about 36 minutes is the one with Baloxavir marboxil RR configuration.
The chromatographic peak with retention time of 16 minutes in fig. 9 is the chromatographic peak with Baloxavir marboxil SR configuration; the 22 min chromatographic peak is that of Baloxavirmarboxil SS configuration.
Figures 8 and 9 demonstrate that the main peak of Baloxavir marboxil and the other 3 configurational impurities are n-hexane in the elution ratio: when ethanol (0.3% tfa, V/V) =80:20, 4 chromatographic peaks can be separated, a condition which can be used for Baloxavir marboxil isomer quality monitoring.
Example 5
Instrument and chromatographic conditions:
Chromatographic column: CHIRALPAK AD-3.150 x 4.6mm,3 μm
A detector: DAD (ultraviolet detector)
Detection wavelength: 210nm;
flow rate: 0.5ml/min;
Column temperature: 35 ℃;
sample injection amount: 5 μl;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=70:30 (V: V)
Run time: 60min;
Diluent/blank solution: ethanol: n-hexane=4:1 (V: V)
The experimental steps are as follows:
about 20mg of Baloxavir marboxil-configuration RS+RR sample is taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale with a diluent, and shaken well to obtain the configuration RS+RR solution.
About 20mg of Baloxavir marboxil-configuration SR+SS sample is taken, precisely weighed into a 10ml brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale, and shaken uniformly to obtain a configuration SR+SS solution.
Taking the configuration RS+RR solution and the configuration SR+SS solution, performing high performance liquid chromatography under the above conditions, and recording chromatograms, wherein the results are shown in FIG. 10 and FIG. 11.
The chromatographic peak with a retention time of 34 minutes in fig. 10 is that of Baloxavir marboxil RS configuration (main compound); the 45 min chromatographic peak is that of Baloxavir marboxil RR configuration.
The chromatographic peak with retention time of 22 minutes in fig. 11 is the chromatographic peak with Baloxavir marboxil SR configuration; the 28 min chromatographic peak is that of Baloxavirmarboxil SS configuration.
Figures 10, 11 demonstrate that Baloxavir marboxil can be separated from its 3 configurational impurities in a mobile phase flow rate of 0.5 ml/min, a condition that can be used for isomer quality monitoring of Baloxavir marboxil.
Example 6
Instrument and chromatographic conditions:
Chromatographic column: CHIRALPAK AD-3.150 x 4.6mm,3 μm
A detector: DAD (ultraviolet detector)
Detection wavelength: 210nm;
flow rate: 0.8ml/min;
column temperature: 38 ℃;
sample injection amount: 5 μl;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=70:30 (V: V)
Run time: for 40min;
Diluent/blank solution: ethanol: n-hexane=4:1 (V: V)
Experimental procedure
About 20mg of Baloxavir marboxil-configuration RS+RR sample is taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale with a diluent, and shaken well to obtain the configuration RS+RR solution.
About 20mg of Baloxavir marboxil-configuration SR+SS sample is taken, precisely weighed into a 10ml brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale, and shaken uniformly to obtain a configuration SR+SS solution.
Taking the configuration RS+RR solution and the configuration SR+SS solution, performing high performance liquid chromatography under the above conditions, and recording chromatograms, wherein the results are shown in FIG. 12 and FIG. 13.
The chromatographic peak with a retention time of about 19 minutes in fig. 12 is the chromatographic peak with the Baloxavir marboxil RS configuration (main compound); the chromatographic peak with a retention time of about 26 minutes is the one with Baloxavir marboxil RR configuration.
The chromatographic peak with a retention time of about 13 minutes in fig. 13 is the chromatographic peak with Baloxavir marboxil SR configuration; the chromatographic peak with retention time of about 17 minutes is the one with Baloxavir marboxil SS configuration.
Figures 12, 13 demonstrate that the Baloxavir marboxil main peak and the other 3 configurational impurities can be separated at column temperature of 38 ℃,4 chromatographic peaks, a condition which can be used for isomer quality monitoring of Baloxavir marboxil.
Example 7
Instrument and chromatographic conditions:
Chromatographic column: CHIRALPAK AD-3.150 x 4.6mm,3 μm
A detector: DAD (ultraviolet detector)
Detection wavelength: 210nm;
flow rate: 0.8ml/min;
Column temperature: 35 ℃;
sample injection amount: 5 μl;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=60:40 (V: V)
Run time: for 40min;
Diluent/blank solution: ethanol: n-hexane=4:1 (V: V)
Experimental procedure
About 20mg of Baloxavir marboxil-configuration RS+RR sample is taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale with a diluent, and shaken well to obtain the configuration RS+RR solution.
About 20mg of Baloxavir marboxil-configuration SR+SS sample is taken, precisely weighed into a 10ml brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale, and shaken uniformly to obtain a configuration SR+SS solution.
Taking the configuration RS+RR solution and the configuration SR+SS solution, performing high performance liquid chromatography under the above conditions, and recording chromatograms, wherein the results are shown in FIG. 14 and FIG. 15.
The chromatographic peak with a retention time of about 13.5 minutes in fig. 14 is the chromatographic peak with the Baloxavir marboxil RS configuration (main compound); the chromatographic peak with retention time of about 17.5 minutes is the one with Baloxavir marboxil RR configuration.
The chromatographic peak with a retention time of about 9 minutes in fig. 15 is the chromatographic peak with Baloxavir marboxil SR configuration; the chromatographic peak with retention time of about 11.5 minutes is the one with Baloxavir marboxil SS configuration.
Fig. 14 and 15 demonstrate that the main peak Baloxavir marboxil and the other 3 configurational impurities are n-hexane in the elution ratio: when ethanol (0.3% tfa) =60:40, 4 chromatographic peaks can be separated, and this condition can be used for isomer quality monitoring of Baloxavir marboxil.
Example 8
Instrument and chromatographic conditions:
Chromatographic column: CHIRALPAK AD-H250 x 4.6mm,5 μm
A detector: DAD (ultraviolet detector)
Detection wavelength: 210nm;
flow rate: 0.8ml/min;
Column temperature: 35 ℃;
sample injection amount: 5 μl;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=70:30 (V: V)
Run time: for 40min;
Diluent/blank solution: ethanol: n-hexane=4:1 (V: V)
Experimental procedure
About 20mg of Baloxavir marboxil-configuration RS+RR sample is taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale with a diluent, and shaken well to obtain the configuration RS+RR solution.
About 20mg of Baloxavir marboxil-configuration SR+SS sample is taken, precisely weighed into a 10ml brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale, and shaken uniformly to obtain a configuration SR+SS solution.
Taking the configuration RS+RR solution and the configuration SR+SS solution, performing high performance liquid chromatography under the above conditions, and recording chromatograms, wherein the results are shown in FIG. 16 and FIG. 17.
The chromatographic peak with a retention time of about 29 minutes in fig. 16 is that of the Baloxavir marboxil RS configuration (main compound); the chromatographic peak of Baloxavirmarboxil RR configuration was not washed out.
The chromatographic peak with a retention time of about 21.5 minutes in fig. 17 is the chromatographic peak with Baloxavir marboxil SR configuration; the chromatographic peak with a retention time of about 28 minutes is the one with Baloxavir marboxil SS configuration.
FIGS. 16, 17 demonstrate that Baloxavir marboxil main peak and other 3 configurational impurities are on the AD-H column, that the RS configuration (main compound) can be separated from its enantiomer (SR), but that the separation of the RS configuration from the SS configuration is poor, and that the RR configuration cannot be washed out in this way. This condition can be used for Baloxavir marboxil enantiomer mass monitoring.
Example 9
Instrument and chromatographic conditions:
Chromatographic column: CHIRALPAK AD 250.4.6 mm,10 μm
A detector: DAD (ultraviolet detector)
Detection wavelength: 210nm;
flow rate: 0.8ml/min;
Column temperature: 35 ℃;
sample injection amount: 5 μl;
Mobile phase a: n-hexane
Mobile phase B: ethanol (0.3% TFA, V/V)
Elution ratio: mobile phase a: mobile phase b=70:30 (V: V)
Run time: 80min;
Diluent/blank solution: ethanol: n-hexane=4:1 (V: V)
Experimental procedure
About 20mg of Baloxavir marboxil-configuration RS+RR sample is taken, precisely weighed into a 10mL brown measuring flask, dissolved and diluted to a scale with a diluent, and shaken well to obtain the configuration RS+RR solution.
About 20mg of Baloxavir marboxil-configuration SR+SS sample is taken, precisely weighed into a 10ml brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale, and shaken uniformly to obtain a configuration SR+SS solution.
Taking the configuration RS+RR solution and the configuration SR+SS solution, performing high performance liquid chromatography under the above conditions, and recording chromatograms, wherein the results are shown in FIG. 18 and FIG. 19.
The chromatographic peaks in figure 18 with a retention time of about 32 minutes are those of the Baloxavir marboxil RS configuration (main compound) and of the configuration RR.
The chromatographic peak with a retention time of about 20 minutes in fig. 19 is the chromatographic peak with Baloxavir marboxil SR configuration; the chromatographic peak with a retention time of about 24 minutes is the one with Baloxavir marboxil SS configuration.
Fig. 18, 19 demonstrate that Baloxavir marboxil main peaks and other 3 configurational impurities are on AD columns, RS configuration (main compound) can be separated from its enantiomer (SR), but RS configuration shares peaks with RR configuration. This condition can be used for Baloxavir marboxil enantiomer mass monitoring.
Claims (5)
1. A method for separating and measuring Baloxavir marboxil and optical isomer thereof by liquid chromatography is characterized in that a chiral chromatographic column with polysaccharide derivative as a filler is adopted, the chiral chromatographic column is CHIRALPAK AD-3, and detection wavelength is 200 nm-230 nm; taking a mixed solution of lower alkane and lower alcohol as a mobile phase; the lower alcohol is ethanol; the lower alkane is n-hexane; the mobile phase contains organic acid; the volume ratio of lower alkane to lower alcohol is 60:40 to 80:20; the volume percentage of the organic acid and the lower alcohol is 0.3 percent.
2. The method according to claim 1, characterized in that: the method comprises the following steps: 1) Taking Baloxavir marboxil or a proper amount of a sample containing Baloxavir marboxil, and dissolving the sample by using a certain amount of a mixed solvent of lower alkane and lower alcohol as a diluent;
2) Setting instrument parameters: the flow rate of the mobile phase, the detection wavelength and the temperature of a chromatographic column incubator;
3) Taking a certain amount of the solution in the step 1), and injecting the solution into a high performance liquid chromatograph to finish Baloxavir marboxil and the separation and determination of the optical isomer thereof.
3. The process of claim 2, wherein the volume ratio of lower alkane to lower alcohol in the diluent is from 50:50 to 10:90.
4. The method according to claim 3, wherein the mixed solvent of lower alkane and lower alcohol is a mixed solvent of n-hexane and ethanol.
5. A process according to claim 3, the mobile phase having a flow rate of 0.5ml/min to 1.5 ml/min; the temperature of the chromatographic column box is 25-40 ℃.
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| Title |
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| Hiroki Koshimichi等.Population Pharmacokinetic and Exposure-Response Analyses of Baloxavir Marboxil in Adults and Adolescents Including Patients With Influenza .《Journal of Pharmaceutical Sciences》.2018,第108卷第1896-1904页. * |
| 陈仲益等.CHIRALPAK AD柱在14种药物手性分离中的应用.《中国药学杂志》.2007,第42卷(第7期),第544-547页. * |
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