CN115266988A - Detection method of related substances of sodium fluridide - Google Patents
Detection method of related substances of sodium fluridide Download PDFInfo
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- CN115266988A CN115266988A CN202210914432.8A CN202210914432A CN115266988A CN 115266988 A CN115266988 A CN 115266988A CN 202210914432 A CN202210914432 A CN 202210914432A CN 115266988 A CN115266988 A CN 115266988A
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 title claims abstract description 52
- 239000011734 sodium Substances 0.000 title claims abstract description 52
- 229910052708 sodium Inorganic materials 0.000 title claims abstract description 52
- 239000000126 substance Substances 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 25
- 239000000243 solution Substances 0.000 claims description 36
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims 1
- 239000002245 particle Substances 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 14
- 239000003814 drug Substances 0.000 abstract description 11
- 238000004458 analytical method Methods 0.000 abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 238000012795 verification Methods 0.000 abstract 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 69
- 239000012535 impurity Substances 0.000 description 45
- 239000012071 phase Substances 0.000 description 19
- 238000007865 diluting Methods 0.000 description 14
- 241000501706 Frasera Species 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000012937 correction Methods 0.000 description 8
- 229940079593 drug Drugs 0.000 description 6
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000000575 pesticide Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 3
- 241000282472 Canis lupus familiaris Species 0.000 description 3
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 3
- 244000045947 parasite Species 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 241000238876 Acari Species 0.000 description 2
- KXDAEFPNCMNJSK-UHFFFAOYSA-N Benzamide Chemical compound NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 description 2
- 108010062745 Chloride Channels Proteins 0.000 description 2
- 102000011045 Chloride Channels Human genes 0.000 description 2
- 241000258922 Ctenocephalides Species 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 2
- QNFBKOHHLAWWTC-UHFFFAOYSA-N Fraxidin Chemical compound C1=CC(=O)OC2=C1C=C(OC)C(OC)=C2O QNFBKOHHLAWWTC-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241001674048 Phthiraptera Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241000258242 Siphonaptera Species 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- ZSWFCLXCOIISFI-UHFFFAOYSA-N cyclopentadiene Chemical compound C1C=CC=C1 ZSWFCLXCOIISFI-UHFFFAOYSA-N 0.000 description 2
- OWZREIFADZCYQD-NSHGMRRFSA-N deltamethrin Chemical compound CC1(C)[C@@H](C=C(Br)Br)[C@H]1C(=O)O[C@H](C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 OWZREIFADZCYQD-NSHGMRRFSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000000749 insecticidal effect Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- -1 (3, 5-dichlorophenyl) -5- (trifluoromethyl) -4, 5-dihydroisoxazol-3-yl Chemical group 0.000 description 1
- ZOCSXAVNDGMNBV-UHFFFAOYSA-N 5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile Chemical compound NC1=C(S(=O)C(F)(F)F)C(C#N)=NN1C1=C(Cl)C=C(C(F)(F)F)C=C1Cl ZOCSXAVNDGMNBV-UHFFFAOYSA-N 0.000 description 1
- OEDUIFSDODUDRK-UHFFFAOYSA-N 5-phenyl-1h-pyrazole Chemical compound N1N=CC=C1C1=CC=CC=C1 OEDUIFSDODUDRK-UHFFFAOYSA-N 0.000 description 1
- 239000005892 Deltamethrin Substances 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 239000005899 Fipronil Substances 0.000 description 1
- YWBVHLJPRPCRSD-UHFFFAOYSA-N Fluridone Chemical compound O=C1C(C=2C=C(C=CC=2)C(F)(F)F)=CN(C)C=C1C1=CC=CC=C1 YWBVHLJPRPCRSD-UHFFFAOYSA-N 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 229960002483 decamethrin Drugs 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 229940013764 fipronil Drugs 0.000 description 1
- MLBZKOGAMRTSKP-UHFFFAOYSA-N fluralaner Chemical compound C1=C(C(=O)NCC(=O)NCC(F)(F)F)C(C)=CC(C=2CC(ON=2)(C=2C=C(Cl)C=C(Cl)C=2)C(F)(F)F)=C1 MLBZKOGAMRTSKP-UHFFFAOYSA-N 0.000 description 1
- 229960004498 fluralaner Drugs 0.000 description 1
- 150000004675 formic acid derivatives Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000171 higher toxicity Toxicity 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 229960003088 loratadine Drugs 0.000 description 1
- JCCNYMKQOSZNPW-UHFFFAOYSA-N loratadine Chemical compound C1CN(C(=O)OCC)CCC1=C1C2=NC=CC=C2CCC2=CC(Cl)=CC=C21 JCCNYMKQOSZNPW-UHFFFAOYSA-N 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 230000037323 metabolic rate Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012224 working solution 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
-
- 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
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
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Abstract
The invention discloses a method for detecting related substances of fluramine sodium, which mainly adopts high performance liquid chromatography to measure and check related substances of a fluramine sodium raw material medicine. The method has high detection rate of related substances, high precision and good repeatability and recovery rate, and can be used for conventional analysis and quality control of the sodium flurandr by verification.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly discloses a drug analysis method for efficiently determining related substances in frasera sodium. The analysis method can effectively separate and measure the related impurities of the frasala sodium and can be used as an effective detection method for controlling the quality of the related substances of the frasala sodium.
Background
Fluramine sodium, english name: fluralaner, chemical name 4- (5 (3, 5-dichlorophenyl) -5- (trifluoromethyl) -4, 5-dihydroisoxazol-3-yl) -2-methyl-N- (2-oxo-2- ((2, 2-trifluoroethyl) amino) ethyl) benzamide. The specific structural formula is shown as follows.
The successful research and development of the frasnailamide creates a brand-new research direction of GABA (gamma-aminobutyric acid) gated chloride ion channel interfering agents, and draws the attention and favor of animal medicine and pesticide science workers. The flurararina mainly plays a role by interfering GABA (Gamma-amino-acid) -gated chloride ion channels, and is similar to the action targets of pesticides such as cyclopentadiene, phenyl pyrazole and macrolide. The flularana is a broad-spectrum pesticide, has good insecticidal activity on pests such as acarina, siphonaptera, phthiraptera, hemiptera, diptera and the like, and has higher toxicity than or equal to that of a common pesticide. The fraxidin has no obvious cross resistance with the existing pesticide, and even has better insecticidal activity to partially resistant pests.
The fluranide has already completed commercial operation as a veterinary drug and is applied to the control of parasites. From 2013 to the present, researchers of German institute of animal health and innovation have carried out a series of experimental studies on fradane, and the prevention and treatment effects of fradane, fipronil (Frontline) and deltamethrin (Scalibor) on dog parasites, ctenocephalides and dermanylus variabilis are comparatively studied by adopting an oral feeding method. The results show that fraserpine has a very good killing effect on ctenocephalides and ticks. In 2014, the merck company is commercially produced for the first time by taking fradora fluoride as a veterinary drug Bravector and is used for killing dog parasites such as lice, fleas and the like. A series of researches show that the medicine can be quickly absorbed by dogs and has the characteristics of quick response, long half-life period, long lasting period, low metabolic rate and the like. The drug has been approved by the FDA in the united states and is sold and popularized in countries such as europe and the united states, and the popularization of the drug in asia and even china is expected to be daily.
In the synthesis process of the frailamide, reaction materials, degradation impurities and process impurities generated in the reaction all affect the quality of the frailamide medicine, the generation of the frailamide impurities needs to be strictly controlled, and a method capable of effectively monitoring related substances of the frailamide medicine needs to be found. Currently in synthesis, the structures of the major impurities produced are as follows:
disclosure of Invention
The invention provides a drug analysis method for efficiently determining related substances in loratadine, which adopts a chromatographic column with octadecylsilane bonded as a filler; taking a buffer salt-organic phase as a mobile phase.
Wherein the buffered salt solution is selected from solutions of: phosphates, formates, acetates, preferably aqueous phosphoric acid solutions; the organic phase is selected from one or a mixture of methanol, ethanol, acetonitrile, propanol and isopropanol, and preferably is a mixed solvent of methanol and acetonitrile.
Preferably, the concentration of the phosphoric acid aqueous solution is 0.05 to 0.5%, preferably 0.1%.
Preferably, the column is a reverse phase column selected from Water, agilent, YMC.
Preferably, the column is of a size of 250 x 4.6,3um, or other equivalent performance column.
Preferably, the column temperature of the method is 35 ℃, and the detection wavelength is 210nm;
preferably, the mobile phase flow rate is between 0.5 and 1.5ml/min, preferably 0.8ml/min.
Preferably, the volume ratio of acetonitrile-methanol is 55-65-35, preferably 50.
Preferably, the method employs gradient elution, in particular, the mobile phase comprises mobile phase a, mobile phase B; the mobile phase A is a sulfuric acid aqueous solution, and the mobile phase B is a mixed solvent of methanol and acetonitrile.
Preferably, the mobile phase of the invention takes and employs the following as table 1:
table 1: gradient of mobile phase
| Time(min) | Mobile Phase A(％) | Mobile Phase B(％) |
| 0 | 33 | 67 |
| 1 | 33 | 67 |
| 40 | 20 | 80 |
| 45 | 5 | 95 |
| 55 | 5 | 95 |
| 55.1 | 33 | 67 |
| 65 | stop |
Furthermore, the invention provides a drug analysis method for efficiently determining related substances in the frasera sodium, which comprises the following steps:
(1) Taking a proper amount of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409, WI-1410 and fluraxel sodium of the fluraxel sodium, adding acetonitrile, ultrasonically dissolving and diluting to prepare a solution containing the known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 of 2.0ug and 1mg of the fluraxel sodium in each 1mL, and shaking up to obtain a system applicability solution.
(2) Taking appropriate amount of fluramine sodium, adding acetonitrile, ultrasonic dissolving, diluting to obtain 1mg of fluramine sodium solution per 1ml, shaking to obtain fluramine sodium sample solution
(3) Precisely measuring a proper amount of the fluranide sodium sample solution, diluting with acetonitrile to a constant volume to prepare a solution containing about 10ug per 1ml, and shaking up to obtain the fluranide sodium reference solution.
(4) The impurity technology adopts a low-concentration external standard method to calculate each impurity, wherein aiming at the prior impurity, a corresponding correction factor needs to be multiplied.
(5) The correction factors for each known impurity are set forth in table 2 below.
Table 2: correction factor for each impurity
| Name(s) | Correction factor |
| WI-1405 | 0.91 |
| WI-1406 | 1.01 |
| WI-1407 | 0.79 |
| WI-1408 | 1.41 |
| WI-1409 | 0.97 |
| WI-1410 | 0.93 |
Description of the drawings:
FIG. 1 is a blank spectrum of the method for detecting related substances in frasera sodium of example 1.
FIG. 2 is a standard working solution spectrum of the method for detecting related substances in frasera sodium of example 1.
FIG. 3 is a special chromatogram of the detection method of related substances of frasera sodium in example 1.
FIG. 4 is a detection limit spectrum of the method for detecting a related substance in frasera sodium of example 1.
FIG. 5 is a quantitative limit chart of the method for detecting related substances in frasera sodium of example 1.
Detailed Description
The method for detecting a substance related to frasera according to the present invention is described in further detail below by way of examples, but it should not be construed that the scope of the subject matter described above is limited to the examples below, and that techniques realized based on the contents described above are within the scope of the present invention.
Example 1: reversed-phase high performance liquid chromatography determination of related substances of fluridone sodium
Measured according to high performance liquid chromatography (appendix VD of four parts of China pharmacopoeia 2020 edition).
The chromatographic condition and system applicability test takes octadecylsilane chemically bonded and silica gel as filling agents; taking 0.1% phosphoric acid water as a mobile phase A; a linear gradient elution was performed using acetonitrile-methanol (50) as mobile phase B according to the following table. The flow rate was 0.8mL per minute and the detection wavelength was 210nm. Taking a proper amount of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409, WI-1410 and fluraxel sodium of the fluraxel sodium, adding acetonitrile, ultrasonically dissolving and diluting to prepare a solution containing the known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 of 2.0ug and 1mg of the fluraxel sodium in each 1mL, and shaking up to obtain a system applicability solution. Precisely measuring 5uL, injecting into a liquid chromatograph, and sequentially peaking off by WI-1408, WI-1409, WI-1406, WI-1405, sodium flurandr, WI-1407 and WI-1410, wherein the separation degree of impurities and sodium flurandr is in accordance with the regulation.
Table 1: mobile phase gradient condition
| Time(min) | Mobile Phase A(％) | Mobile Phase B(％) |
| 0 | 33 | 67 |
| 1 | 33 | 67 |
| 40 | 20 | 80 |
| 45 | 5 | 95 |
| 55 | 5 | 95 |
| 55.1 | 33 | 67 |
| 65 | stop |
The determination method comprises taking appropriate amount of the product, adding acetonitrile, ultrasonic dissolving, diluting to obtain 1mg solution containing fluoride sodium Railamide per 1ml, shaking to obtain sample solution; precisely measuring appropriate amount, diluting with acetonitrile to desired volume to obtain solution containing about 10ug per 1ml, and shaking to obtain control solution. Injecting 5ul of the contrast solution into a liquid chromatograph, and adjusting the detection sensitivity to make the full-scale range of the high-defect recorder of the chromatographic peak of the main component 10-25%. Precisely measuring 5ul of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, recording a chromatogram, deducting a solvent peak from the impurity peak in the chromatogram of the test solution, calculating the relative retention time of each impurity by taking the frasera sodium peak as a reference, and substituting into the correction factor of each impurity in the following table for calculation. Wherein the peak area of each impurity is not more than one fifth of the main peak area (0.2%) of the control solution, the peak area of other single impurity is not more than one fifth of the main peak area (0.2%) of the control solution, and the sum of the peak areas of each impurity is not more than the main peak area (1%) of the control solution. Correction factors for each impurity are as in table 2:
table 2: correction factor for each impurity
| Name (R) | Correction factor |
| WI-1405 | 0.91 |
| WI-1406 | 1.01 |
| WI-1407 | 0.79 |
| WI-1408 | 1.41 |
| WI-1409 | 0.97 |
| WI-1410 | 0.93 |
1. Experiment of system applicability: the chromatographic condition and system applicability test takes octadecylsilane chemically bonded and silica gel as filling agents; linear gradient elution was performed with 0.1% phosphoric acid water as mobile phase a and acetonitrile-methanol (50) as mobile phase B according to the following table. The flow rate was 0.8mL per minute and the detection wavelength was 210nm. Taking a proper amount of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409, WI-1410 and fluraxel sodium of the fluraxel sodium, adding acetonitrile, ultrasonically dissolving and diluting to prepare a solution containing the known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 of 2.0ug and 1mg of the fluraxel sodium in each 1ml, and shaking up to obtain a system applicability solution. Precisely measuring 5uL, injecting into a liquid chromatograph, and sequentially peaking off by WI-1408, WI-1409, WI-1406, WI-1405, sodium flurandr, WI-1407 and WI-1410, wherein the separation degree of impurities and sodium flurandr is in accordance with the regulation.
2. The specificity is as follows: the separation performance of the proposed chromatographic conditions was verified by known and potential impurity configurations. The experimental results are as follows: precisely measuring 5ul of system applicability solution, injecting into a liquid chromatograph, and obtaining experimental results as shown in table 3:
table 3: specificity results
| Peak | RT(min) | RRT | Resolution |
| WI-1408 | 17.396 | 0.58 | NA |
| WI-1409 | 18.089 | 0.60 | 1.4 |
| RRT0.74 | 22.392 | 0.74 | 7.5 |
| WI-1406 | 24.955 | 0.83 | 4.2 |
| WI-1405 | 25.796 | 0.86 | 1.5 |
| Frirana | 30.114 | 1.00 | 7.2 |
| WI-1407 | 33.288 | 1.11 | 5.0 |
| RRT1.15 | 34.6 | 1.15 | 2.0 |
| WI-1410 | 40.434 | 1.34 | 8.6 |
| RRT1.78 | 53.483 | 1.78 | 24.6 |
The separation degree of the frasera sodium and adjacent impurities is 7.2 and 5.0, and the separation degree of the known impurities is more than 1.2, so that the method meets the requirement of specificity.
3. Linearity and range: taking a proper amount of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409, WI-1410 and fluramine sodium of the fluramine, adding acetonitrile, ultrasonically dissolving and diluting to prepare a solution containing 100ug of each of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 and 100ug of fluramine sodium per 1mL, shaking up to be used as a linear standard stock solution; precisely measuring 0.1ml, 0.2ml, 0.4ml, 1.0ml and 2.0ml to 20ml volumetric flasks, adding acetonitrile to dilute to a scale, and preparing linear solutions L1-L5; precisely measuring 5ul, recording peak area, drawing a standard curve by taking the peak area A as a vertical coordinate and the sample concentration C as a horizontal coordinate, and calculating a regression equation. The results are shown in Table 4:
all linearity results met the linearity requirement, with the linear concentration range as shown in table 4:
table 4: main material and each impurity linearity conclusion
| Identification | Concentration of | Relative concentration level |
| Frirana | 0.5019μg/mL～10.0370μg/mL | 0.05％～1.0％ |
| WI-1405 | 0.4961μg/mL～9.9221μg/mL | 0.05％～1.0％ |
| WI-1406 | 0.4417μg/mL～8.8347μg/mL | 0.05％～1.0％ |
| WI-1407 | 0.4558μg/mL～9.1161μg/mL | 0.05％～1.0％ |
| WI-1408 | 0.5003μg/mL～10.0062μg/mL | 0.05％～1.0％ |
| WI-1409 | 0.5008μg/mL～10.0152μg/mL | 0.05％～1.0％ |
| WI-1410 | 0.4581μg/mL～9.1620μg/mL | 0.05％～1.0％ |
4. Accuracy: taking a proper amount of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409, WI-1410 and fluraxel sodium of the fluraxel sodium, adding acetonitrile, ultrasonically dissolving and diluting to prepare solutions containing 100ug of each of the known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 and 1mg of the fluraxel sodium in every 1mL, shaking up to be used as a recovery standard stock solution; precisely measuring 0.1ml, 0.4ml and 2.0ml to 20ml volumetric flasks, adding acetonitrile to dilute to a scale, and preparing recovery solutions RM-L1, RM-L3 and RM-L5; precisely measure 5ul, and record peak area.
The results are shown in tables 5 to 11:
table 5: control sample results
| Label | WI-1405 | WI-1406 | WI-1407 | WI-1408 | WI-1409 | WI-1410 |
| Control Sample-1 | ND | ND | ND | ND | ND | ND |
| Control Sample-2 | ND | ND | ND | ND | ND | ND |
| Control Sample-3 | ND | ND | ND | ND | ND | ND |
Table 6: WI-1405 recovery results
Table 7: WI-1406 recycle results
Table 8: WI-1409 recovery results
Table 9: WI-1408 recovery results
Table 10: WI-1409 recovery results
Table 11: WI-1410 recovery of results
The experimental results are as follows: tables 6-11 show that the quantitative limit recovery of impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 is 80.8-120.7%, the RSD% recovery of the three-needle is 1.8-6.3%, the limit recovery of 100% and 500% is 95.3-107.4%, and the RSD% recovery is 0.46-2.7%, which meets the requirement of accuracy.
4. Precision:
(1) repeatability: taking a proper amount of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409, WI-1410 and fluraxel sodium of the fluraxel sodium, adding acetonitrile, ultrasonically dissolving and diluting to prepare solutions containing 100ug of each of the known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 and 1mg of the fluraxel sodium in each 1ml, shaking up to obtain a standard recovery stock solution; precisely measuring 0.4ml to 20ml volumetric flasks, adding acetonitrile to dilute to a scale, and preparing a recovered solution RM-L3; measure 5ul precisely and record the peak area. The results are shown in Table 12:
table 12: repetitive results
The experimental results are as follows: table 12 shows that the RSD% of 6 known impurities is 1.0-2.5%, the absolute difference of unknown impurities is 0.01%, and the repeatability requirement is met.
(2) Intermediate precision: taking a proper amount of the product by a determination method, adding acetonitrile, ultrasonically dissolving and diluting to prepare a solution containing 1mg of the fluoride-containing sodium ralfate in 1ml, and shaking up to be used as a test solution; precisely measuring appropriate amount, diluting with acetonitrile to desired volume to obtain solution containing about 10ug per 1ml, and shaking to obtain control solution. Taking a proper amount of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409, WI-1410 and fluraxel sodium of the fluraxel sodium, adding acetonitrile, ultrasonically dissolving and diluting to prepare solutions containing 100ug of each of the known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 and 1mg of the fluraxel sodium in each 1ml, shaking up to obtain a standard recovery stock solution; precisely measuring 0.4ml to 20ml volumetric flasks, adding acetonitrile to dilute to a scale, and preparing a recovered solution RM-L3; precisely measure 5ul, and record peak area.
Table 13: intermediate precision results
The experimental results are as follows: the system applicability meets the requirements of the system. Table 13 shows that different analyzers use different instruments to perform experiments on different dates, and that the RSD values of the contents of 12 samples (detection results of two analyzers) of WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 are respectively 2.2% -4.9% and the absolute difference value of unknown impurities is 0.01% under the limit of 100%, so that the requirement of precision is met.
5. Sensitivity: taking a proper amount of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409, WI-1410 and fluraxel sodium of the fluraxel sodium, adding acetonitrile, ultrasonically dissolving and diluting to prepare solutions containing 100ug of known impurities WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 and 100ug of fluraxel sodium in each 1ml of the solution, and shaking up to be used as standard stock solutions of sensitivity; precisely measuring a 0.1ml to 50ml volumetric flask, adding acetonitrile to dilute to a scale, and preparing a sensitivity solution DL1; precisely measuring a 0.1ml to 20ml volumetric flask, adding acetonitrile to dilute to a scale, and preparing a sensitivity solution DL2; precisely measure 5ul, and record peak area.
The experimental results show that: the detection limits of Frirana, WI-1405, WI-1406, WI-1407, WI-1408, WI-1409 and WI-1410 are respectively 0.02 percent, and the signal-to-noise ratios of the two are all larger than 3; the quantitative limit is 0.05 percent, the signal to noise ratios are all more than 10, and the RSD of the retention time of each impurity of 6 continuous needles is 0.77 to 1.2 percent; RSD of the peak area is 1.9% -7.8%; the requirement of sensitivity is met.
6. Durability:
(1) changing the column temperature
(2) Change post
Table 14: durability results
The experimental results are as follows: as can be seen from table 14, when the C18 chromatographic columns of different lot numbers are replaced, the column temperature is within the ± 2 ℃ variation range, the system applicability meets the requirements, and the detection results of the related substances have no obvious change, and the analysis method meets the requirements on durability within the variation range.
Claims (7)
1. A detection method for measuring related substances of fraseral sodium is characterized in that: the method adopts a chromatographic column which adopts octadecylsilane bonded as a filler; taking a phosphoric acid water mobile phase as an A phase and taking an acetonitrile-methanol mixed solution mobile phase as a B phase; wherein the concentration of the sulfuric acid aqueous solution is between 0.05 percent and 0.5 percent, the volume ratio of acetonitrile-methanol is between 45 and 65 to 35, the certain flow rate is between 0.5 ml/min and 1.5ml/min, the certain column temperature condition is set to be between 30 ℃ and 45 ℃, and the following gradient elution is adopted:
。
2. the method of claim 1, wherein the chromatographic column has a particle size of 3.0um, a column length of 250mm, and a column inner diameter of 4.6mm.
3. The method of claim 1, wherein the concentration of the aqueous phosphoric acid solution is 0.05% to 0.5%.
4. The method of claim 1, wherein the concentration of the aqueous phosphoric acid solution is selected from 0.05% or 0.1%.
5. The method according to claim 1, wherein the volume ratio of acetonitrile-methanol in the mobile phase B is 55-65-35.
6. The method according to claim 1, wherein the flow rate of the mobile phase in the method is 0.5 to 1.5ml/min.
7. The method of claim 1, wherein the column temperature is between 30 ℃ and 45 ℃, the detection wavelength is 210nm, and the sample volume is 5uL.
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