CN115327005B - Method for detecting clopidogrel oxide related substances - Google Patents
Method for detecting clopidogrel oxide related substances Download PDFInfo
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- CN115327005B CN115327005B CN202210964672.9A CN202210964672A CN115327005B CN 115327005 B CN115327005 B CN 115327005B CN 202210964672 A CN202210964672 A CN 202210964672A CN 115327005 B CN115327005 B CN 115327005B
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- 238000000034 method Methods 0.000 title claims abstract description 76
- 239000000126 substance Substances 0.000 title claims abstract description 58
- 239000005552 B01AC04 - Clopidogrel Substances 0.000 title claims abstract description 18
- 229960003009 clopidogrel Drugs 0.000 title claims abstract description 18
- GKTWGGQPFAXNFI-HNNXBMFYSA-N clopidogrel Chemical compound C1([C@H](N2CC=3C=CSC=3CC2)C(=O)OC)=CC=CC=C1Cl GKTWGGQPFAXNFI-HNNXBMFYSA-N 0.000 title abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 87
- 239000000243 solution Substances 0.000 claims abstract description 71
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 238000010828 elution Methods 0.000 claims abstract description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000012266 salt solution Substances 0.000 claims abstract description 17
- 239000012488 sample solution Substances 0.000 claims abstract description 15
- 239000002904 solvent Substances 0.000 claims abstract description 12
- 230000002378 acidificating effect Effects 0.000 claims abstract description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000945 filler Substances 0.000 claims abstract description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims abstract description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000010790 dilution Methods 0.000 claims abstract description 3
- 239000012895 dilution Substances 0.000 claims abstract description 3
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 239000013558 reference substance Substances 0.000 claims description 14
- 239000011259 mixed solution Substances 0.000 claims description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- HRQDCDQDOPSGBR-UHFFFAOYSA-M sodium;octane-1-sulfonate Chemical compound [Na+].CCCCCCCCS([O-])(=O)=O HRQDCDQDOPSGBR-UHFFFAOYSA-M 0.000 claims description 11
- 238000007865 diluting Methods 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- 150000002500 ions Chemical class 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 8
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 8
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 5
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 4
- 239000005695 Ammonium acetate Substances 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 229960000583 acetic acid Drugs 0.000 claims description 4
- 235000019257 ammonium acetate Nutrition 0.000 claims description 4
- 229940043376 ammonium acetate Drugs 0.000 claims description 4
- 239000012362 glacial acetic acid Substances 0.000 claims description 4
- 239000012046 mixed solvent Substances 0.000 claims description 4
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 4
- ROBLTDOHDSGGDT-UHFFFAOYSA-M sodium;pentane-1-sulfonate Chemical compound [Na+].CCCCCS([O-])(=O)=O ROBLTDOHDSGGDT-UHFFFAOYSA-M 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- 125000003158 alcohol group Chemical group 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 2
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 2
- 235000011056 potassium acetate Nutrition 0.000 claims description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000001632 sodium acetate Substances 0.000 claims description 2
- 235000017281 sodium acetate Nutrition 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 claims description 2
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 claims description 2
- QWSZRRAAFHGKCH-UHFFFAOYSA-M sodium;hexane-1-sulfonate Chemical compound [Na+].CCCCCCS([O-])(=O)=O QWSZRRAAFHGKCH-UHFFFAOYSA-M 0.000 claims description 2
- 239000003002 pH adjusting agent Substances 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 7
- 238000004458 analytical method Methods 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 description 117
- 239000012071 phase Substances 0.000 description 80
- 239000000523 sample Substances 0.000 description 23
- 238000012360 testing method Methods 0.000 description 22
- 229940126062 Compound A Drugs 0.000 description 19
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 description 19
- 238000010606 normalization Methods 0.000 description 18
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- 238000002347 injection Methods 0.000 description 11
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- 238000011084 recovery Methods 0.000 description 8
- 238000010812 external standard method Methods 0.000 description 7
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- 238000004364 calculation method Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 230000007423 decrease Effects 0.000 description 5
- 238000003908 quality control method Methods 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- RWOLDZZTBNYTMS-SSDOTTSWSA-N (2r)-2-(2-chlorophenyl)-2-hydroxyacetic acid Chemical compound OC(=O)[C@H](O)C1=CC=CC=C1Cl RWOLDZZTBNYTMS-SSDOTTSWSA-N 0.000 description 1
- 125000004182 2-chlorophenyl group Chemical group [H]C1=C([H])C(Cl)=C(*)C([H])=C1[H] 0.000 description 1
- JXRGUPLJCCDGKG-UHFFFAOYSA-N 4-nitrobenzenesulfonyl chloride Chemical compound [O-][N+](=O)C1=CC=C(S(Cl)(=O)=O)C=C1 JXRGUPLJCCDGKG-UHFFFAOYSA-N 0.000 description 1
- 241000244987 Daiswa polyphylla Species 0.000 description 1
- 101000783577 Dendroaspis angusticeps Thrombostatin Proteins 0.000 description 1
- 101000783578 Dendroaspis jamesoni kaimosae Dendroaspin Proteins 0.000 description 1
- 206010017472 Fumbling Diseases 0.000 description 1
- 229940127218 antiplatelet drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 150000002085 enols Chemical class 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- IEMGWBMVQLVHEY-UHFFFAOYSA-N ethyl 2-(3-amino-6,7-dihydro-5h-cyclopenta[b]pyridin-7-yl)acetate Chemical compound NC1=CN=C2C(CC(=O)OCC)CCC2=C1 IEMGWBMVQLVHEY-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- QWXYZCJEXYQNEI-OSZHWHEXSA-N intermediate I Chemical compound COC(=O)[C@@]1(C=O)[C@H]2CC=[N+](C\C2=C\C)CCc2c1[nH]c1ccccc21 QWXYZCJEXYQNEI-OSZHWHEXSA-N 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
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- 230000007935 neutral effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
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- 239000000106 platelet aggregation inhibitor Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Abstract
The invention relates to the field of drug detection and analysis, in particular to a method for detecting clopidogrel oxide related substances. The method for detecting the clopidogrel oxide related substances adopts a high performance liquid chromatography, takes (7 aS,2' S) -2-oxygen-clopidogrel and obtains a sample solution after solvent dilution; injecting the sample solution into a high performance liquid chromatograph, and detecting according to chromatographic conditions; the chromatographic condition uses octadecylsilane chemically bonded silica as filler, the detection wavelength is 220+ -2 nm, the salt solution with acidic pH value containing acidic ion pair is used as mobile phase A, the solution containing acetonitrile and/or alcohol is used as mobile phase B, and gradient elution is performed. The detection method provided by the invention has good specificity, high sensitivity and high accuracy, and is suitable for researching the registration quality of the (7 aS,2' S) -2-oxo-clopidogrel medicine.
Description
Technical Field
The invention relates to the field of drug detection and analysis, in particular to a detection method of (7 aS,2' S) -2-oxygen-clopidogrel related substances.
Background
(7 aS, 2's) -2-oxo-clopidogrel (hereinafter referred to aS "compound a"), a metabolite of clopidogrel in the human body, is a more active and safer platelet aggregation inhibitor, and has the chemical name: methyl (S) -2- (2-chlorophenyl) -2- ((S) -2-oxo-2, 6,7 a-tetrahydrothiophene [3,2-c ] pyridin-5 (4H) yl) acetate:
。
the current preparation process of the compound A mainly adopts a route reported in a patent CN104245707A, R- (-) -o-chloromandelic acid (SM 1) is taken as a starting material, an intermediate I is generated after methyl esterification, a key intermediate II is generated by condensation of p-nitrobenzenesulfonyl chloride (SM 2), and the target compound A is generated after substitution and purification. The general reaction route is as follows:
。
according to the molecular characteristics and the preparation process of the compound A, related substances mainly comprise degradation impurities, isomers and process impurities. Wherein the degradation impurities such as hydroxyl impurity (P1Z 5), enol impurity (P1Z 12), major isomer such as P1Z2, and key process impurity intermediate II (P1Z 1), the structural details of each impurity are shown in Table 1.
。
The research on the substances is important to the quality control of medicines, but the prior art has not completely reported information about the quality control of the compound A, and particularly the research on the quality of the substances. Although the specification of the prior art (CN 111943958A) discloses a detection method for calibrating a compound of formula I, the method cannot meet the requirement of simultaneously detecting the above impurities, and particularly cannot meet the requirement of detecting process impurities. In order to better control related substances of the compound A, meet the requirement of medicine marketing, develop a related substance quality control method suitable for the compound A, and solve the technical problem in the research of the medicine quality at present.
Disclosure of Invention
In order to solve the quality control requirement of (7 aS,2 'S) -2-oxygen-clopidogrel, the invention provides a detection method of (7 aS,2' S) -2-oxygen-clopidogrel related substances.
The method for detecting the (7 aS,2' S) -2-oxygen-clopidogrel related substances can detect most impurities including but not limited to the impurities P1Z1, P1Z2, P1Z5 and P1Z12 by adopting a high performance liquid chromatography.
The invention provides a method for detecting (7 aS,2' S) -2-oxygen-clopidogrel related substances, which adopts high performance liquid chromatography and comprises the following operation steps:
(1) Taking (7 aS,2' S) -2-oxygen-clopidogrel, and diluting with a solvent to obtain a sample solution;
(2) And (3) injecting the sample solution into a high performance liquid chromatograph, and detecting by adopting chromatographic conditions.
In certain embodiments, the chromatographic conditions described above comprise: the chromatographic column with octadecylsilane chemically bonded silica as filler has detection wavelength of 220+ -2 nm, salt solution with acidic pH value containing acidic ion pair as mobile phase A, and solution containing acetonitrile and/or alcohol as mobile phase B, and is eluted in gradient.
In certain embodiments, the gradient elution described above comprises at least two procedures: the proportion of the mobile phase A in the first section program is 35-60%, and the proportion of the mobile phase B is 65-40%; the proportion of the mobile phase A in the second section program is 5-20%, and the proportion of the mobile phase B is 95-80%; and the total time of the first section of program is 10-50 minutes, and the total time of the second section of program is 10-50 minutes.
Further, the total time of the second program includes a proportional decrease time of the mobile phase A, a proportional increase time of the mobile phase B, and a maintaining time, wherein the proportional decrease time of the mobile phase A is equal to the proportional increase time of the mobile phase B, and is 5-30 minutes, and the maintaining time is 5-30 minutes.
In certain embodiments, the above detection method comprises a system applicability test: and (3) respectively weighing the impurity reference substance and a proper amount of (7 aS,2 'S) -2-oxygen-clopidogrel, and dissolving and diluting the impurity reference substance and the (7 aS,2' S) -2-oxygen-clopidogrel to a mixed solution with a target concentration by using a solvent in a disposable preparation mode or a mode of firstly concentrating and then diluting.
Further, the system applicability test adopts a mode of firstly preparing the mixture in a concentrated mode and then preparing the mixture in a diluted mode: respectively taking a proper amount of impurity reference substances, dissolving and quantitatively diluting the impurity reference substances by using a solvent to prepare mixed solutions which contain 5-250 ug in each 1ml, taking the mixed solutions aS impurity stock solution, additionally taking a proper amount of (7 aS,2 'S) -2-oxo-clopidogrel, placing the mixed solutions into a measuring flask, adding a proper amount of the impurity stock solution, dissolving and quantitatively diluting the mixed solutions by using the solvent to prepare 0.5-10 ug of each impurity in each 1ml, (7 aS,2' S) -2-oxo-clopidogrel, and preparing 0.5-10 mg of each impurity.
In certain embodiments, the gradient elution procedure for the chromatographic conditions described above comprises: the proportion of the mobile phase A is 35-60% in 0 min, and the proportion of the mobile phase B is 65-40% and is maintained for 20-40 min; the proportion of the mobile phase A is reduced to 5-20% in 30-60 min, and the mobile phase B is increased to 95-80% and maintained for 50-70 min.
In certain embodiments, the gradient elution procedure for the chromatographic conditions described above comprises: the proportion of the mobile phase A is 35-60% in 0 min, and the proportion of the mobile phase B is 65-40% and is maintained for 25-35 min; the proportion of the mobile phase A is reduced to 5-20% in 40-60 min, and the mobile phase B is increased to 95-80% and maintained for 55-65 min.
Further, the gradient elution procedure of the above chromatographic conditions comprises: the proportion of the mobile phase A is 35-60% in 0 min, and the proportion of the mobile phase B is 65-40% and is maintained for 30 min; the proportion of mobile phase A decreases to 5% -20% at 50 min, and mobile phase B increases to 95% -80% and is maintained for 60 min.
Further, the gradient elution procedure of the above chromatographic conditions comprises: the proportion of mobile phase A was 46% and the proportion of mobile phase B was 54% at 0 min and maintained for 30 min; at 50 minutes the proportion of mobile phase a was reduced to 10% and mobile phase B was raised to 90% and maintained for 60 minutes.
In certain embodiments, the gradient elution procedure described above further comprises a system balancing procedure that includes a recovery time and a maintenance time.
Further, the recovery time is 0.1 to 20 minutes, preferably 0.1 to 5 minutes; the holding time is 0.1 to 20 minutes, preferably 10 minutes.
Further, the gradient elution procedure further includes a system balancing procedure: the proportion of the mobile phase A rises to 35% -60% and the proportion of the mobile phase B falls to 65% -40% in 50.1-71 minutes, and the mobile phase A is maintained for 5-20 minutes.
Further, the gradient elution procedure described above also includes a system equilibration procedure, with an exemplary increase in mobile phase A ratio of 35% to 60% and a decrease in mobile phase B ratio of 65% to 40% for 61 minutes, and maintained for 70 minutes.
Still further, the system balancing procedure described above, for example, increases the proportion of mobile phase a to 46% and decreases the proportion of mobile phase B to 54% at 61 minutes and maintains it for 70 minutes.
In certain embodiments, the chromatographic conditions described above have a flow rate of 0.5 to 2.0ml/min; preferably 0.8 to 1.5ml/min.
In certain embodiments, the column temperature for the chromatographic conditions described above is from 0 to 40 ℃; preferably 20 to 40 ℃.
In certain embodiments, the related substances comprise one or more of P1Z5, P1Z2, P1Z1, P1Z12.
In some embodiments, the solvent comprises acetonitrile, THF, alcohol or a mixed solvent of mobile phases B and a in a volume ratio of 40:60 to 100:0; acetonitrile is preferred.
Further, the solvent is a mixed solvent of mobile phases B and A in a volume ratio of 50:50-95:5, 55:45, 60:40, 65:35, 80:20, 85:15, or 90:10.
In certain embodiments, the acidic ion pairs comprise sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, sodium octanesulfonate, or sodium dodecanesulfonate.
Further, the concentration of the acidic ion pair is 0.001mol/L to 2.0mol/L; preferably 0.005mol/L to 0.5mol/L.
In certain embodiments, the salt of the salt solution comprises potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, sodium acetate, or potassium acetate.
Further, the concentration of the salt solution is 0.001mol/L to 2.0mol/L; preferably 0.005mol/L to 0.5mol/L.
In some embodiments, the pH of the salt solution is 1.0 to 6.0; preferably 2.0 to 5.0.
Further, the pH value is regulated by a pH regulator, wherein the pH regulator comprises phosphoric acid, hydrochloric acid, sulfuric acid, glacial acetic acid or formic acid.
In some embodiments, the mobile phase B is alcohol, acetonitrile or a mixed solution of alcohol and acetonitrile in a volume ratio of 80:20-100:0; preferably, the volume ratio of the alcohol to the acetonitrile is 90:10-98:2.
Further, the above alcohol includes methanol, ethanol, propanol or isopropanol.
In some embodiments, the mobile phase A is a salt solution (pH 1.0-6.0) containing sodium octane sulfonate. The definition of the salt solution is the same as above. Further, the pH is preferably 2.0 to 5.0.
Further, the mobile phase A is a salt solution (pH value is 1.0-6.0) containing 0.001 mol/L-2.0 mol/L of sodium octane sulfonate solution. The definition of the salt solution is the same as above.
Further, the mobile phase A is a salt solution (pH 1.0 to 6.0) containing 0.001mol/L to 2.0mol/L of sodium octane sulfonate solution. The definition of the salt solution is the same as above.
Further, the mobile phase A is a potassium dihydrogen phosphate solution (pH value is 1.0-6.0) containing 0.001 mol/L-2.0 mol/L of sodium octane sulfonate solution.
In certain embodiments, mobile phase B is acetonitrile.
In certain embodiments, mobile phase B is an alcohol. Alcohols are as defined above.
In some embodiments, the mobile phase B is a mixed solution of acetonitrile and alcohol in a volume ratio of 80:20-100:0; preferably, the volume ratio of the alcohol to the acetonitrile is 90:10-98:2. Alcohols are as defined above.
In certain embodiments, the system suitability test described above peaks in the following order: P1Z5, P1Z2, compound A, P Z1, P1Z12.
In certain embodiments, the chromatogram of the test solution includes the following peaks in the following order: P1Z5, P1Z2, compound A, P Z1, P1Z12.
In certain embodiments, the content of the relevant substance in the chromatogram of the test solution is calculated as a peak area by an external standard method and/or calculated as a peak area by a self-contrast method and/or calculated as a peak area normalization method.
In certain embodiments, the impurities P1Z1 and P1Z5 do not exceed 0.1% in the chromatogram of the test solution, if any; and/or the impurity P1Z2 is not more than 1.0%; and/or the impurity P1Z12 is not more than 0.1%; and/or other single unknown impurities of no more than 0.1%; and/or total impurities of not more than 2.0%.
In certain embodiments, the impurity P1Z1 and P1Z5 in the chromatogram of the test solution, if related substances are related, are calculated by an external standard method according to peak area, and the peak area is not more than 0.1%; and/or impurity P1Z2 is not more than 1.0% as calculated by peak area normalization; and/or impurity P1Z2 is not more than 1.0% as calculated by peak area by self-contrast; and/or the calculation of other single impurities according to a peak area normalization method is less than 0.1%; and/or other individual impurities are less than 0.1% calculated as peak area by self-contrast.
In certain embodiments, the impurity P1Z1 and P1Z5 in the chromatogram of the test solution, if related substances are related, are calculated by an external standard method according to peak area, and the peak area is not more than 0.1%; and/or impurity P1Z2 is not more than 1.0% as calculated by peak area normalization; and/or the other individual impurities are less than 0.1% calculated by peak area normalization.
In some embodiments, the chromatogram of the sample solution contains no more than 2.0% of the sum of peak areas of the impurities, as calculated by peak area normalization.
Advantageous effects: the method for detecting the (7 aS,2' S) -2-oxygen-clopidogrel related substance has the advantages of good peak type of each peak, good separation degree of each peak being more than 1.5, good method specificity, high sensitivity and accuracy, meets the quality analysis and detection requirements, and is suitable for researching the quality of related substances of the product.
Drawings
Fig. 1: system applicability solution chromatograms.
Detailed Description
The scheme of the present invention will be explained below in conjunction with experimental examples and examples. Those skilled in the art will appreciate that the following examples are illustrative of the present invention only and should not be construed as limiting the scope of the invention. The specific techniques or conditions not identified in each example are according to the techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products obtained commercially without the manufacturer's attention.
Compound a was dissolved in acetonitrile, slightly soluble in methanol, and almost insoluble in water. The working control (lot number: 201113-D, purity 99.6%) and test (lot number: 39201001) of Compound A used in the following experimental examples and examples were all derived from Chengdu Shi Beikang biomedical technology Co., ltd, and the P1Z5 control was purchased from TRC; the isomer impurity P1Z2 and the impurity P1Z12 cannot be synthesized due to instability, but can be detected in the main medicine, and are matched with LC-MS detection and positioning.
Experimental example 1: methodology investigation of compound A related substance detection method
Preparation of various reference solutions and test solutions of herba Cynanchi Paniculati
Test solution: a proper amount of the sample (Compound A, lot number: 39201001) was taken, precisely weighed, dissolved in acetonitrile and quantitatively diluted to prepare a solution (available as new) containing about 1mg per 1 ml.
Impurity control solution: the P1Z5 reference substance and the P1Z1 reference substance are taken respectively in proper amounts, dissolved by acetonitrile and quantitatively diluted to prepare mixed solutions with about 1 mug each 1 ml.
System applicability solution: taking appropriate amounts of P1Z5 reference substance and P1Z1 reference substance respectively, dissolving with acetonitrile, quantitatively diluting to obtain mixed solution containing about 10ug each 1ml as impurity stock solution, taking about 10mg of compound A, placing into a 10ml measuring flask, precisely adding 1ml of impurity stock solution, diluting with acetonitrile to scale, and shaking.
Description: the solution can also be prepared by dissolving with mixed solvents including but not limited to THF, alcohol or mobile phases B and A in a volume ratio of 40:60-100:0, and other reagents which can dissolve the product.
Chromatographic conditions of Paris polyphylla
According to the chemical structural formula of the compound A, the inventor prefers chromatographic conditions disclosed in the specification test 1 of the patent CN111943958A, but when the impurity P1Z1 is added, the impurity P1Z1 is overlapped with the impurity P1Z12, and the detection method of related substances of the product is finally determined by fumbling, adding an ion pair reagent, and then matching with optimization modes such as adjusting a gradient elution program.
(1) Selection of mobile phase and gradient elution procedure
Under otherwise unchanged conditions, the mobile phase was screened as follows:
(a) Mobile phase a: 0.01mol/L potassium dihydrogen phosphate solution (pH value adjusted to 3.0 by phosphoric acid) containing 0.01mol/L sodium octane sulfonate solution, mobile phase B: methanol;
(b) Mobile phase a: 0.01mol/L potassium dihydrogen phosphate solution (pH value adjusted to 3.0 by phosphoric acid) containing 0.01mol/L sodium octane sulfonate solution, mobile phase B: methanol: acetonitrile (94:6, v/v);
(c) Mobile phase a: 0.01mol/L potassium dihydrogen phosphate solution (pH value adjusted to 3.0 by phosphoric acid) containing 0.01mol/L sodium octane sulfonate solution, mobile phase B: acetonitrile.
Gradient elution procedure 1 is as described in Table 1-1:
。
the gradient elution procedure is as described in tables 1-2:
。
and (3) adopting chromatographic conditions a, gradient elution procedures 1-2, chromatographic conditions b and c and gradient elution procedure 2 to sample and detect the system applicability solution respectively, and collecting chromatograms.
The results illustrate: the chromatographic condition a and the gradient elution procedure 1 have good peak patterns and poor separation degree between impurities. Under the chromatographic condition a and the gradient elution procedure 2, the peak type is good, the main peak and each impurity can be well separated, and the impurity peaks can be well separated. Under the conditions of the chromatographic conditions b and c and the gradient elution procedure 2, the peak types are good, the main peak and each impurity can be well separated, and the impurity peaks can be well separated.
(2) Selection of mobile phase aqueous phase pH
Under the condition that other conditions are not changed, the pH of seven mobile phases A is selected for testing, and any one of phosphoric acid, hydrochloric acid, sulfuric acid, glacial acetic acid or formic acid is adopted as a pH regulator, namely pH1 (1.0), pH2 (2.0), pH3 (3.0), pH4 (4.0), pH5 (5.0), pH6 (6.0) and pH7 (7.0). By adopting the conditions, sample injection detection is respectively carried out on the system applicability solution, and a chromatogram is acquired.
The results illustrate: under the first six pH values, no matter what pH regulator is adopted, the peak type is good, the main peak and each impurity can be well separated, the impurity peaks can also be well separated, the separation degree of each peak is more than 1.5, and the measured known impurity content and impurity number have no obvious change. However, under neutral conditions of pH7.0, part of the impurities cannot be separated effectively, and part of the impurity peaks are asymmetric in peak form. Indicating that the pH value of the acid mobile phase has no influence on the measurement result of related substances.
(3) Column temperature selection
Under other conditions, four column temperatures were selected for the test, column temperature 1 (0 ℃), column temperature 2 (20 ℃), column temperature 3 (30 ℃) and column temperature 4 (40 ℃). By adopting the conditions, sample injection detection is respectively carried out on the system applicability solution, and a chromatogram is acquired.
The results illustrate: under four column temperature conditions, the peak type is good, the main peak and each impurity can be well separated, the impurity peaks can also be well separated, the separation degree of each peak is more than 1.5, and the measured known impurity content and the impurity number have no obvious change. Indicating that the column temperature has no influence on the measurement result of the related substances.
(4) Selection of flow rate
Under other conditions, four chromatographic flow rates were selected for the test, 1 (0.5 ml/min), 2 (1.0 ml/min), 3 (1.5 ml/min) and 4 (2.0 ml/min). And adopting the four conditions to sample and detect the solution with the system applicability respectively, and collecting the chromatograms.
The results illustrate: under four flow rate conditions, the peak type is good, the main peak and each impurity can be well separated, the impurity peaks can also be well separated, the separation degree of each peak is more than 1.5, and the measured known impurity content and the impurity number have no obvious change. Indicating that the flow rate has no effect on the measurement of the relevant substances.
(5) Selection of chromatographic column model
Under other conditions, three different chromatographic column models were selected: model 1: c18 column, 250 x 4.6mm,5 μm, model 2: c18 column, 250 x 4.6mm,3.5 μm, model 3: c18 column, 250 x 3.0mm,3.0 mu m, sample injection detection to system adaptability solution respectively, and chromatogram collection.
The results illustrate: under the conditions of three types, the peak types are good, the main peak and each impurity can be well separated, the impurity peaks can also be well separated, the separation degree of each peak is more than 1.5, and the measured known impurity content and the impurity number have no obvious change. Indicating that the model of the C18 column has no influence on the measurement result of related substances. In addition, the inventors basically tried common C18 columns, which can meet the requirement of the separation degree of more than 1.5, and each peak type is good, and prove that the chromatographic method of the invention is not influenced by the type and the brand of the column.
Experimental example 2: methodological verification
Verification of specificity of the heart
Chromatographic conditions: the detection wavelength is 220nm, and the mobile phase and gradient elution procedure is mobile phase C and gradient elution procedure 3 in Experimental example 1, with a flow rate of 1.0ml/min and a column temperature of 30 ℃, using a chromatographic column (Chromcore 120C 18.6 mm. Times.250 mm 5 μm) with octadecylsilane bonded silica gel as the filler.
The preparation method of each solution is the same as that of experimental example 1.
Taking the known impurities P1Z1 and P1Z5 and the compound A in proper amounts, and carrying out sample injection measurement according to the established chromatographic conditions. The analysis results are shown in Table 2-1, and the chromatogram of the system applicability solution is shown in FIG. 1.
The test result shows that under the condition of the planned chromatograph, the solvent does not interfere with the detection of each impurity in the sample solution, and the separation degree between the impurities and the main peak is more than 1.5; the peak purity index average meets the analysis requirement; the peak sequence of each substance is as follows: P1Z5, P1Z2, main peaks, P1Z1 and P1Z12, and the result shows that the method has good specificity.
Strong breaking test of the capsule
The preparation method of the sample comprises the following steps: weighing 3 parts of a proper amount of compound A sample (batch number: 39201001), respectively dissolving the sample with 2ml of acetonitrile, respectively adding 0.1ml of 0.1mol/L hydrochloric acid or 70ul of 0.1mol/L sodium hydroxide or 1ml of 3% hydrogen peroxide, standing the mixture at room temperature for 2 minutes with strong acid and strong alkali, standing the mixture at the room temperature for 24 hours with oxidation, respectively dissolving and diluting the mixture with acetonitrile after strong power failure to prepare a solution with about 1mg per 1 ml. The results of the brute force failure test are set forth in Table 2-2.
Under the conditions of forced destructive testing of acid, alkali and oxidation, the separation degree of the main peak and the adjacent peaks is more than 1.5, the detection of known impurities is not interfered by degraded impurities, and the purity parameters of the main peak meet the regulations.
⒊ detection line and quantitative limit
Taking a proper amount of each of impurities P1Z5 and P1Z1 and a compound A working reference substance, respectively adding acetonitrile for dissolution and stepwise dilution, and taking the detection limit of each substance when the peak height of the substance is about 3 times of the baseline noise, namely the signal-to-noise ratio is about 3:1; the limit of quantification of this substance is when the peak height is about 10 times the baseline noise, i.e., the signal to noise ratio is about 10:1. The results are shown in tables 2-3.
The test results show that: the detection limit of the impurity P1Z5 is 1.080ng, the detection limit of the other impurities is lower than that of the P1Z5, the sample injection concentration planned by the related substance inspection is 1mg/ml, the sample injection amount is 10 mu l, and the absolute sample injection amount is 10000ng. The absolute sample injection amount is more than 9000 times of the P1Z5 detection limit, which shows that the formulated sample injection concentration can ensure the effective detection of the impurities of the sample.
⒋ linearity and range
Taking appropriate amounts of the reference substance stock solutions of the impurities P1Z5 and P1Z1 under the 'special' test item, precisely measuring, adding acetonitrile to dilute to prepare 10%, 20%, 50%, 80%, 100% and 200% solutions with the limiting concentration, taking 1 μl of each of the linear reference substance solutions according to the established chromatographic conditions, injecting into a gas chromatograph, recording the chromatograms, carrying out linear regression by taking the concentration as an abscissa and the peak area as an ordinate, and obtaining the results shown in tables 2-4.
In conclusion, when the concentration of P1Z5 is between 0.1002 mug/ml and 2.0040 mug/ml, the linear relation between the concentration and the peak area is good; the concentration of the impurity P1Z1 is in the range of 0.1080-2.1600 mug/ml, and the concentration and the peak area are in good linear relation; the concentration of the compound A is in the range of 0.1169-2.3380 mug/ml, and the concentration and the peak area of the compound A have good linear relation; the ratio of Y-axis intercept to 100% peak area is within 25%.
⒌ precision and repeatability test
Taking a reference substance solution mixed with 100% concentration impurities under the linear test item, continuously sampling for 6 times, recording peak area, and examining sampling precision. The results show that: the RSD of the impurity P1Z5 peak area is: RSD of 0.9% for impurity P1Z1 peak area: 2.1 percent, which all meet the requirements, meets the requirement of liquid chromatography precision measurement.
Preparing 6 parts of test sample solution in parallel, testing the same sample injection precision of the control sample solution, measuring the content of each impurity in the test sample according to the established chromatographic conditions, calculating the impurity content of the impurities P1Z5 and P1Z1 according to an external standard method and the peak area, and calculating the impurity content of the impurities P1Z2 and P1Z12 and the total impurities according to a self-comparison method and an area normalization method respectively, wherein the results are shown in tables 2-5.
From the above results, it was found that the impurities P1Z5 and P1Z1 were not detected in the 6 samples, and the impurities P1Z2 and P1Z12 were detected, and the detection results of the impurities P1Z2 and P1Z12 and the total impurities in the 6 samples were calculated by the self-comparison method and the area normalization method, and the RSD of the 6 samples by the area normalization method was better than the self-comparison method, so that the area normalization method was selected as the calculation method of the impurities. 6 samples are detected by an area normalization method, the average value of impurities P1Z2 is 0.25%, the average value of P1Z12 is 0.02%, the average value of RSD is 17.0%, the average value of total impurities is 0.27%, and the RSD is 1.6%, which shows that the method has good repeatability.
⒍ recovery rate
In order to examine the degree of approach between the detection results and the true values of the impurities P1Z5 and P1Z1 by the proposed detection method, the accuracy is verified by adopting a sample adding and recycling mode. Taking a proper amount of a test sample with known impurity content, preparing a test sample solution according to a formulated method, respectively adding known impurities with limit concentrations of 80%, 100% and 120% into the test sample solution, and then carrying out sample injection measurement to calculate the recovery rate. The test results are shown in tables 2-6.
From the above results, the recovery rate of the P1Z5 sample is 97.36% -105.60%, the average recovery rate is 102.4%, and the RSD is 2.6%; the recovery rate of the P1Z1 sample is 91.12% -102.08%, the average recovery rate is 97.2%, and the RSD is 3.2%, which shows that the method can accurately detect P1Z5 and P1Z1 impurities in the product, and has good accuracy.
⒎ durability
In order to verify the degree to which the accuracy of the measurement results is not affected when the conditions of the developed liquid phase slightly change, the durability of the developed chromatographic conditions was examined.
And (3) taking a system applicability solution, a sample solution and a self control solution, and measuring according to the established chromatographic conditions.
The test results show that when the flow rate, the column temperature, the chromatographic column and the mobile phase slightly change, such as the flow rate change of +/-0.2 ml/min, the column temperature change of +/-5 ℃, the change of chromatographic columns of different types (the packing is unchanged), the proportion change of the mobile phase B of +/-2 and the pH value change of the fluidity A of +/-2, the measured known impurity content and the impurity number have no obvious change under all conditions. The column temperature, the flow rate, the chromatographic column and the pH value of the mobile phase have no influence on the measurement result of related substances.
Example 1: examination of substances 1 (Mobile phase 1)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the mobile phase B was methanol under chromatographic conditions, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 2: examination of substances 2 (Mobile phase 2)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the mobile phase B was acetonitrile under chromatographic conditions, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 3: determination of substances of interest 3 (Mobile phase 3)
The preparation and chromatographic conditions of each solution are the same as those of experimental example 2, wherein the mobile phase B of the chromatographic conditions is a mixed solution of methanol and acetonitrile in a volume ratio of 80:20, and the other conditions are unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 4: determination of substances of interest 4 (Mobile phase 4)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the acidic ion pair in the mobile phase A of the chromatographic conditions was changed from 0.01mol/L sodium octane sulfonate to 2.0mol/L sodium pentane sulfonate, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 5: determination of substances of interest 5 (Mobile phase 5)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the salt solution in mobile phase A of chromatographic conditions was changed from 0.01mol/L of potassium dihydrogen phosphate to 0.001mol/L of ammonium acetate, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 6: determination of substances of interest 6 (Mobile phase 6)
The solution formulation and chromatographic conditions were the same as in experimental example 2, with mobile phase a adjusted to pH 5.0 with glacial acetic acid, the other conditions being unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 7: determination of substances of interest 7 (gradient procedure 3)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the gradient elution procedure is shown in Table 3-1, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 8: determination of substances of interest 8 (gradient procedure 4)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the gradient elution procedure is shown in Table 3-2, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 9: determination of substances of interest 9 (gradient program 5)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the gradient elution procedure is shown in tables 3-3, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 10: determination of substances of interest 10 (gradient procedure 6)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the gradient elution procedure is shown in tables 3 to 4, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 11: determination of substances of interest 11 (gradient program 7)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the gradient elution procedure is shown in tables 3 to 5, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 12: determination of substances of interest 12 (gradient program 8)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the gradient elution procedure is shown in tables 3 to 6, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 13: determination of substances of interest 13 (pH of mobile phase A1.0)
The preparation and chromatographic conditions of each solution are the same as in experimental example 2, wherein the pH value of the mobile phase A is changed from original 4.0 to 1.0, and the other conditions are unchanged.
And (3) measuring: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 14: determination of substances of interest 14 (pH of Mobile phase A6.0)
The preparation and chromatographic conditions of each solution are the same as in experimental example 2, wherein the pH value of the mobile phase A is changed from 4.0 to 6.0, and the other conditions are unchanged.
And (3) measuring: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 15: determination of substances of interest 15
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the flow rate was changed from 1.0ml/min to 2.0ml/min, and the other conditions were unchanged.
And (3) measuring: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 16: related substance measurement 16
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the flow rate was changed from 1.0ml/min to 0.5ml/min, and the other conditions were unchanged.
And (3) measuring: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 17: determination of substances of interest 17 (Mobile phase 7)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the acidic ion pair in the mobile phase A of the chromatographic conditions was changed from 0.01mol/L sodium octane sulfonate to 0.001mol/L sodium pentane sulfonate, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
Example 18: determination of substances 18 (Mobile phase 8)
The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the salt solution in mobile phase A of chromatographic conditions was changed from 0.01mol/L of potassium dihydrogen phosphate to 2.0mol/L of ammonium acetate, and the other conditions were unchanged.
Assay: the solutions were injected into the chromatographic system and the chromatograms were collected.
It is noted that the last 10 minutes of examples 1 to 10, 13 to 18 and the last 20 minutes of examples 11 to 12 described above are system equilibrium times, and that this part of the procedure no longer has impurity elution peaks.
The impurities P1Z1 and P1Z5 are calculated according to an external standard method and the peak areas of the impurities P1Z2 and P1Z12 are calculated according to a self-comparison method or an area normalization method, and other single impurities and total impurities are calculated according to the peak area normalization method.
Data statistics and summary: summarizing experimental example 2 under the specific term and the respective chromatograms of examples 1 to 18 above, the peak area, retention time and degree of separation were counted, and the results showed that: (1) in each system applicability solution chromatogram: the peak type of each peak is better, the separation degree between each main peak and impurity peak and between impurity peak and impurity peak is between 1.5 and 15.0, both are more than 1.5, the separation degree of each peak is better, and each method has good exclusive property and meets the quality analysis requirement. (2) in each sample solution chromatogram: the difference between the content of each main peak compound A and the content of each experimental example 2 calculated by the peak area according to an external standard method is within 0.1 percent; the impurities P1Z5 and P1Z1 are not detected, the impurities P1Z2 and P1Z12 are detected, the content of the impurities P1Z2 is 0.24-0.27% by calculation through a self-comparison method and an area normalization method, the content of the impurities P1Z12 is 0.01-0.04%, and the total impurities are not more than 0.30% by calculation through the area normalization method, so that the accuracy and the reproducibility of the proposed related substance measuring method are good. According to the method, the quality control standard of the product can be determined, namely, if impurity peaks exist in the sample solution, the impurities P1Z1 and P1Z5 are calculated according to an external standard method and calculated according to peak areas, and the peak areas are not more than 0.1%; the impurity P1Z2 is not more than 1.0% calculated by a peak area calculation or a peak area normalization method according to a self-comparison method; the calculation of other single unknown impurities according to a peak area normalization method is less than 0.1%; the sum of the peak areas of the impurities is not more than 2.0%. Wherein the impurity P1Z12 is not more than 0.1% calculated by a peak area method or calculated by an area normalization method according to a self-comparison method; can be controlled according to unknown impurities.
Claims (12)
1. A method for detecting (7 aS, 2's) -2-oxy-clopidogrel and related substances, which is characterized in that the method adopts high performance liquid chromatography, and comprises the following steps:
taking (7 aS,2' S) -2-oxygen-clopidogrel, and diluting with a solvent to obtain a sample solution;
injecting the sample solution into a high performance liquid chromatograph, and detecting according to chromatographic conditions;
system applicability solution: respectively weighing the related substance reference substance and a proper amount of (7 aS,2 'S) -2-oxygen-clopidogrel, and dissolving and diluting the related substance reference substance and the (7 aS,2' S) -2-oxygen-clopidogrel to a mixed solution with a target concentration by using a solvent in a disposable preparation mode or a first concentration and then dilution mode;
injecting the system applicability solution into a high performance liquid chromatograph, and detecting according to chromatographic conditions;
the chromatographic conditions include: octadecylsilane chemically bonded silica is used as a filler, the detection wavelength is 220+/-2 nm, a salt solution with an acidic pH value containing acidic ion pairs is used as a mobile phase A, a solution containing acetonitrile and/or alcohol is used as a mobile phase B, and gradient elution is carried out; the gradient elution procedure included: the proportion of the mobile phase A is 35-60% in 0 min, and the proportion of the mobile phase B is 65-40% and is maintained for 20-40 min; the proportion of the mobile phase A is reduced to 5-20% in 30-60 min, and the mobile phase B is increased to 95-80% and maintained for 50-70 min;
the related substances comprise two or more of P1Z1, P1Z2, P1Z5 and P1Z12, wherein the structures of the P1Z1, the P1Z2, the P1Z5 and the P1Z12 are respectively as follows:
。
2. the method according to claim 1, wherein the solvent comprises acetonitrile, THF, alcohol or a mixed solvent of mobile phases B and a in a volume ratio of 40:60 to 100:0.
3. The method of claim 2, wherein the solvent is acetonitrile.
4. The method of claim 1, wherein the acidic ion pair comprises sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, sodium octanesulfonate, or sodium dodecanesulfonate.
5. The method according to claim 1, wherein the salt of the salt solution comprises potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, sodium acetate, or potassium acetate.
6. The method according to claim 1, wherein the pH of the salt solution is 1.0 to 6.0.
7. The method according to claim 6, wherein the pH of the salt solution is 2.0 to 5.0.
8. The method of claim 1, wherein the pH is adjusted with a pH adjuster comprising phosphoric acid, hydrochloric acid, sulfuric acid, glacial acetic acid, or formic acid.
9. The detection method according to claim 1, wherein the mobile phase B is alcohol, acetonitrile or a mixed solution of alcohol and acetonitrile in a volume ratio of 80:20 to 100:0.
10. The method according to claim 9, wherein the mobile phase B is a mixed solution of alcohol and acetonitrile in a volume ratio of 90:10 to 98:2.
11. The method according to any one of claims 1 to 10, wherein the alcohol comprises methanol, ethanol, propanol or isopropanol.
12. The method according to any one of claims 1 to 10, wherein the chromatogram of the sample solution contains, if any, not more than 0.1% of P1Z1 and P1Z 5; and/or P1Z2 is not more than 1.0%; and/or P1Z12 is not more than 0.1%; and/or other individual unknown related substances not exceeding 0.1%; and/or the total relevant substances do not exceed 2.0%.
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| Identification of alcohol-dependent clopidogrel metabolites using conventional liquid chromatography/triple quadrupole mass spectrometry;Zhe-Yi Hu等;Rapid Commun. Mass Spectrom(第28期);1285-1292 * |
| Method development and validation for Clopidogrel bisulphate and its related substances by Fast LC;Ramakant Sharma等;Journal of Pharmacy Research;第4卷(第10期);3351-3354 * |
| STRUCTURE AND STEREOCHEMISTRY OF THE ACTIVE METABOLITE OF CLOPIDOGREL;JEAN-MARIE PEREILLO等;DRUG METABOLISM AND DISPOSITION;第30卷(第11期);1288–1295 * |
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