CN105699524B - The detection method of isomer impurities content in a kind of ticagrelor - Google Patents
The detection method of isomer impurities content in a kind of ticagrelor Download PDFInfo
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- CN105699524B CN105699524B CN201610070436.7A CN201610070436A CN105699524B CN 105699524 B CN105699524 B CN 105699524B CN 201610070436 A CN201610070436 A CN 201610070436A CN 105699524 B CN105699524 B CN 105699524B
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Abstract
The invention discloses a kind of detection methods of isomer impurities content in ticagrelor, it includes the following steps：A, reference substance solution is prepared；B, test solution is prepared；C, reference substance solution, test solution are detected respectively using efficient liquid-phase chromatography method；D, by external standard method the isomer impurities content in ticagrelor sample is obtained with calculated by peak area or according to the standard curve calculating between peak area and concentration.The present invention provides a kind of new detection methods of isomer impurities content in ticagrelor, separating degree between each chromatographic peak is high, it is noiseless between each other, it can realize simultaneously to impurity B and impurity A, the accurate detection of impurity C, content for isomer impurities in monitoring ticagrelor drug provides a kind of effective detection method, further ensures the product quality of ticagrelor and the drug safety of patient.
Description
Technical field
The invention belongs to the analysis detection fields in chemistry, and in particular to isomer impurities content in a kind of ticagrelor
Detection method.
Background technology
Ticagrelor (Ticagrelor trade names：BRILINTA), entitled (1S, 2S, 3R, the 5S) -3- of chemistry [7- [[(1R,
2S) -2- (3,4- difluorophenyls) cyclopropyl] amino] -5- rosickyite base -3H-1,2,3- triazolyls [4,5-d] pyrimidin-3-yl] -5-
(2- hydroxy ethoxies) -1,2- pentamethylene glycol (molecular formula：C23H28F2N6O4S), it is by AstraZeneca pharmaceutical Co. Ltd
A kind of selective adenosine diphosphate (ADP) (ADP) receptor antagonist of (AstraZeneca AB) exploitation, by activating P2Y12By
Body, the platelet activation of reversible retardance ADP mediations and aggregation.
Due to being limited by production technology and controlled level, in the ticagrelor product for producing gained, often there are more
Kind impurity, for example, (1R, 2R, 3S, 5R) -3- [7- { [(1R, 2S) -2- (3,4- difluorophenyl) cyclopropyl] amino } -5- rosickyite
Base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol, (1R, 2R, 3S,
5R) -3- [7- { [(1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl] amino } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d]
Pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol, (1S, 2S, 3R, 5S) -3- [7- { [(1S, 2R) -2- (3,4-
Difluorophenyl) cyclopropyl] amino } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies)
Pentamethylene -1,2- glycol etc. (referring to：Ticagrelor impurity list,http://wenku.baidu.com/view/ 69885bd8fd0a79563c1e72aa.htmlRe=view, the time of disclosure is on October 17th, 2014), it seriously affects for lattice
The product quality of auspicious Lip river and the drug safety of patient.
However, for detecting ticagrelor, an only patent document CN 104634887A is had detected for lattice at present
A kind of impurity content in auspicious Lip river：(1S, 2S, 3R, 5S) -3- (7- ((1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl amino) -5-
(rosickyite base) -3H- [1,2,3] triazole [4,5-d] pyrimidin-3-yl) -5- (2- hydroxyl-oxethyls) pentamethylene -1,2- glycol, it is difficult to
Comprehensively reflect the product quality of ticagrelor.
In order to be detected to impurity more in ticagrelor, meanwhile, also for more fully monitoring ticagrelor
Product quality is further ensured that the drug safety of patient, needs to invent a kind of new detection method.
Invention content
The purpose of the present invention is to provide a kind of detection methods of isomer impurities content in ticagrelor.
The detection method of isomer impurities content in a kind of ticagrelor provided by the invention, it includes the following steps：
A, the reference substance of isomer impurities is taken, prepares reference substance solution；
B, ticagrelor sample to be checked is taken, prepares test solution；
C, reference substance solution, test solution are detected respectively using efficient liquid-phase chromatography method, the efficient liquid
The testing conditions of phase chromatographic process are：
Chromatographic column：Stationary phase is amylose-three (3,5- xylyls carbamate)；
Mobile phase：The volume ratio of n-hexane-ethanol solution, n-hexane and ethyl alcohol is 88%：12%~92%：8%, it is described
Ethyl alcohol in the diethylamine containing 0.1%~0.5%ml/ml；
Detection wavelength：200nm~300nm；
D, it is replaced by external standard method with calculated by peak area or according to the standard curve calculating between peak area and concentration
Isomer impurities content in Ge Ruiluo samples.
Further, the isomer impurities include at least (1R, 2R, 3S, 5R) -3- [7- { [(1R, 2S) -2- (3,4-
Difluorophenyl) cyclopropyl] amino } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies)
Pentamethylene -1,2- glycol.
Further, the isomer impurities include at least (1R, 2R, 3S, 5R) -3- [7- { [(1R, 2S) -2- (3,4-
Difluorophenyl) cyclopropyl] amino } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies)
Pentamethylene -1,2- glycol and (1R, 2R, 3S, 5R) -3- [7- { [(1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl] amino } -5-
Rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol；
Preferably, the isomer impurities are simultaneously including (1R, 2R, 3S, 5R) -3- [7- { [(1R, 2S) -2- (3,4- bis-
Fluorophenyl) cyclopropyl] amino } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) ring
Pentane -1,2- glycol, (1R, 2R, 3S, 5R) -3- [7- { [(1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl] amino } -5- third
Sulfenyl -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol and (1S, 2S, 3R,
5S) -3- [7- { [(1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl] amino } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d]
Pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol.
Further, in step a, the solvent for preparing reference substance solution is the mobile phase in step c；In step b, prepare and supply
The solvent of test sample solution is the mobile phase in step c.
Further, in step c, the chromatographic column isAD-H, length 250mm, internal diameter are
4.6mm, the grain size of filler is 5 μm.
Further, in step c, the column temperature of the chromatographic column is 25 DEG C~35 DEG C.
Further, in step c, the volume ratio of n-hexane and ethyl alcohol is 90%：10%；Contain in the ethyl alcohol
The diethylamine of 0.2%ml/ml.
Further, in step c, the flow velocity of the mobile phase is 0.9ml/min~1.1ml/min；Preferably, the stream
The flow velocity of dynamic phase is 1.0ml/min.
Further, in step c, the Detection wavelength is 230nm~250nm；Preferably, the Detection wavelength is
242nm。
Further, in step c, sampling volume is the μ l of 10 μ l~50；Preferably, sampling volume is 20 μ l.
The present invention provides a kind of new detection method of isomer impurities content in ticagrelor, dividing between each chromatographic peak
It is high from degree, it is noiseless between each other, it can realize simultaneously to impurity B and impurity A, the accurate detection of impurity C, moreover, operation letter
Just, it is easy to control, testing cost is low, and with good linear relationship, specificity, precision, stability, sensitivity and repetition
Property, sample recovery rate is high, and testing result is accurate, reliable, and the content for isomer impurities in monitoring ticagrelor drug provides
A kind of effective detection method, further ensures the product quality of ticagrelor and the drug safety of patient.
Obviously, the above according to the present invention according to the ordinary technical knowledge and customary means of this field, is not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Description of the drawings
Fig. 1 is the HPLC figures of impurity A reference substance solution under testing conditions of the present invention.
Fig. 2 is the HPLC figures of impurity B reference substance solution under testing conditions of the present invention.
Fig. 3 is the HPLC figures of impurity C reference substance solutions under testing conditions of the present invention.
Fig. 4 is the HPLC figures of impurity A under testing conditions of the present invention, impurity B, impurity C mixed solutions.
Fig. 5 is the HPLC figures of ticagrelor test solution under testing conditions of the present invention.
Fig. 6 be ticagrelor and impurity A under testing conditions of the present invention, impurity B, impurity C mixed solutions HPLC scheme.
Fig. 7 is the HPLC figures of impurity A reference substance solution under 1 chromatographic condition of contrast test.
Fig. 8 is the HPLC figures of impurity B reference substance solution under 1 chromatographic condition of contrast test.
Fig. 9 is the HPLC figures of impurity C reference substance solutions under 1 chromatographic condition of contrast test.
Figure 10 is the HPLC figures of ticagrelor test solution under 1 chromatographic condition of contrast test
Figure 11 is the HPLC figures of impurity A reference substance solution under 2 chromatographic condition of contrast test.
Figure 12 is the HPLC figures of impurity B reference substance solution under 2 chromatographic condition of contrast test.
Figure 13 is the HPLC figures of impurity C reference substance solutions under 2 chromatographic condition of contrast test.
Figure 14 is the HPLC figures of ticagrelor test solution under 2 chromatographic condition of contrast test
Figure 15 is the HPLC figures of poly-doped impurity solution under 2 chromatographic condition of contrast test
Figure 16 is the HPLC figures of impurity A reference substance solution under 3 chromatographic condition of contrast test.
Figure 17 is the HPLC figures of impurity B reference substance solution under 3 chromatographic condition of contrast test.
Figure 18 is the HPLC figures of impurity C reference substance solutions under 3 chromatographic condition of contrast test.
Figure 19 is the HPLC figures of ticagrelor test solution under 3 chromatographic condition of contrast test.
Figure 20 is the HPLC figures of poly-doped impurity solution under 3 chromatographic condition of contrast test.
Figure 21 is that the mixed solution HPLC of ticagrelor and impurity schemes under 3 chromatographic condition of contrast test.
Figure 22 is the ultraviolet spectrogram of the 1st lower ticagrelor of test example 1.
Figure 23 is the canonical plotting of the 2nd lower impurity A reference substance of test example 1, and ordinate is peak area, and abscissa is dense
Degree.
Figure 24 is the canonical plotting of the 2nd lower impurity B reference substance of test example 1, and ordinate is peak area, and abscissa is dense
Degree.
Figure 25 is the canonical plotting of the 2nd lower impurity C reference substance of test example 1, and ordinate is peak area, and abscissa is dense
Degree.
Specific embodiment
The raw material that is used in the specific embodiment of the invention, equipment are known product, are obtained by buying commercial product.
Embodiment 1,
The raw material that is used in the specific embodiment of the invention, equipment are known product, are obtained by buying commercial product；
For example, the lot number of ticagrelor is 20140801；Impurity A lot number is 20140213, content：99.6%；Impurity B batch
Number for 20140227, content：98.4%；Impurity C lot numbers are 20140307, content：99.1%；Derive from hundred abundant science and technology of Chengdu
Pharmaceutical Co. Ltd.
Entitled (1R, 2R, 3S, 5R) -3- [7- { [(1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl] ammonia of impurity A
Base } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol.
Entitled (1R, 2R, 3S, 5R) -3- [7- { [(1R, 2S) -2- (3,4- difluorophenyls) cyclopropyl] ammonia of impurity B
Base } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol.
Entitled (1S, 2S, 3R, 5S) -3- [7- { [(1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl] ammonia of impurity C
Base } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol.
AUW220D type precision electronic balances are purchased from Shimadzu Corporation；LC-20AT type efficient liquid-phase chromatographic pumps are purchased from Shimadzu
Company, LC-20AT type efficient liquid-phase chromatographic pumps are purchased from Shimadzu Corporation, and SPD-M20A DAD detectors are purchased from Shimadzu Corporation,
SIL-20A autosamplers are purchased from Shimadzu Corporation, and LcSolution work stations are purchased from Shimadzu Corporation；1525 types of Waters
Efficient liquid-phase chromatographic pump is purchased from water generation company,AD-H (250mm × 4.6mm, 5 μm) chromatographic column is commercially available
From Daicel company；Agilent ZorbaxC18 (250mm × 4.6mm, 3.5 μm) are purchased from Agilent Co., Ltd.
The efficient liquid-phase chromatography method of chiral isomer impurity in embodiment 1, present invention detection ticagrelor
Chromatographic column：AD-H, 4.6mm × 250mm, 5 μm；
Mobile phase：N-hexane-ethyl alcohol (90:10) diethylamine of 0.1%~0.5%ml/ml, is added in ethyl alcohol
Solvent：Mobile phase
Column temperature：30℃；Flow velocity：1.0ml/min；UV detectors (Detection wavelength 242nm).
Sampling volume：20μL.
Detecting step：
It takes impurity A, B and C reference substances appropriate, is dissolved with solvent, be configured to the reference substance solution of the impure each 5 μ g of every 1mL.
Take ticagrelor sample appropriate, after being dissolved with ethyl alcohol, solubilization dilution agent is configured to every 1mL containing about 0.5mg for examination
Product solution.
It takes ticagrelor appropriate, after being dissolved with ethyl alcohol, adds in impurity A, B and C reference substance solutions, diluted with solvent, prepare
Into mixed solution.
Measuring method：Each 20 μ L injections liquid chromatograph of above-mentioned solution is taken, chromatogram is recorded, as a result such as Fig. 1~Fig. 6.
Fig. 1 is that the HPLC of impurity A reference substance solution schemes, and the retention time of impurity A is 65.553min.
Fig. 2 is that the HPLC of impurity B reference substance solution schemes, and the retention time of impurity B is 42.595min.
Fig. 3 is that the HPLC of impurity C reference substance solutions schemes, and the retention time of impurity C is 53.713min.
Fig. 4 is that the HPLC of poly-doped impurity reference substance solution schemes, and the retention time of impurity A is 70.675min, the guarantor of impurity B
The time is stayed as 45.761min, the retention time of impurity C is 58.058min.
Fig. 5 is that the HPLC of test solution schemes, and the retention time of ticagrelor main peak is 40.045min.
Fig. 6 is the HPLC figures of test sample and impurity A, impurity B and impurity C mixed solutions, and the retention time of ticagrelor is
39.658min, the retention time of impurity B are 45.346min, and the retention time of impurity C is 57.591min, during the reservation of impurity A
Between for 70.185min, the separating degree between ticagrelor and impurity B and impurity is respectively 2.688,5.153,4.472.
The result shows that under chromatographic condition of the invention, the separating degree between ticagrelor and impurity is high, can realize for lattice
It is detected while a variety of chiral impurity contents in auspicious Lip river.
Contrast test 1：
Chromatographic column：Agilent Zorbax C184.6mm × 250mm, 3.5 μm；
Mobile phase A：1. phosphate buffer (takes 1.0mol/L sodium dihydrogen phosphates (with phosphoric acid tune pH value to 3.0)
10ml adds water to 900ml)-acetonitrile (90:10)；
Mobile phase B：2. phosphate buffer (takes 1.0mol/L sodium dihydrogen phosphates (with phosphoric acid tune pH value to 3.0)
10ml adds water to 300ml)-acetonitrile (30:70)；
Solvent：Acetonitrile-water (50:50)
Column temperature：35℃；Flow velocity：1.0ml/min；UV detectors (Detection wavelength 242nm).
Condition of gradient elution is as follows：
It takes impurity A reference substance appropriate, is dissolved with solvent, be configured to impurity A reference substance solutions of every 1mL containing about 0.5mg.
Take impurity B appropriate, solubilizer is configured to impurity B reference substance solutions of every 1mL containing about 0.5mg.
Take impurity C appropriate, solubilizer is configured to impurity C reference substance solutions of every 1mL containing about 0.5mg.
Take ticagrelor appropriate, solubilizer is configured to test solutions of every 1mL containing about ticagrelor 0.5mg.
Measuring method：Above-mentioned 20 μ L of solution injections liquid chromatograph is taken, chromatogram is recorded, as a result as shown in Fig. 7~10.
Fig. 7 is the HPLC figures of impurity A reference substance solution under 1 chromatographic condition of contrast test, and impurity A retention time is
29.493min。
Fig. 8 is that the HPLC of impurity B reference substance solution under 1 chromatographic condition of contrast test schemes, the retention time of impurity B
29.450min。
Fig. 9 is the HPLC figures of impurity C reference substance solutions under 1 chromatographic condition of contrast test, and the retention time of impurity C is
29.461min。
Figure 10 is that the HPLC of test solution under 1 chromatographic condition of contrast test schemes, the retention time of ticagrelor
29.457min。
The result shows that almost indistinction, this method completely cannot for impurity A, impurity B, impurity C and ticagrelor retention time
Carry out impurity A, impurity B and impurity C separation detections.
Contrast test 2：
Chromatographic column：OD-H, 4.6mm × 250mm, 5 μm；
Mobile phase：N-hexane-ethyl alcohol (70:30)
Solvent：Mobile phase
Column temperature：30℃；Flow velocity：1.0ml/min；UV detectors (Detection wavelength 242nm).
Sampling volume：20μL.
It takes impurity A reference substance appropriate, is dissolved with solvent, be configured to impurity A reference substance solutions of every 1mL containing about 0.5 μ g.
It takes impurity B reference substance appropriate, is dissolved with solvent, be configured to impurity B reference substance solutions of every 1mL containing about 10 μ g.
It takes impurity C reference substances appropriate, is dissolved with solvent, be configured to impurity C reference substance solutions of every 1mL containing about 20 μ g.
It takes ticagrelor sample appropriate, is dissolved with solvent, be configured to test solutions of every 1mL containing about 20 μ g.
Take ticagrelor and impurity A, impurity B, impurity C reference substance solutions appropriate, solubilizer is diluted to every 1ml containing each ingredient
The mixed solution of 0.5 μ g.
Measuring method：Above-mentioned 20 μ L of solution injections liquid chromatograph is taken, chromatogram is recorded, as a result as shown in Figure 11~17.
Figure 11 is the HPLC figures of impurity A reference substance solution under 2 chromatographic condition of contrast test, and the retention time of impurity A is
12.500min。
Figure 12 is the HPLC figures of impurity B reference substance solution under 2 chromatographic condition of contrast test, and the retention time of impurity B is
9.796min。
Figure 13 is the HPLC figures of impurity C reference substance solutions under 2 chromatographic condition of contrast test, and the retention time of impurity C is
11.189min。
Figure 14 is the HPLC figures of ticagrelor test solution under 2 chromatographic condition of contrast test, when ticagrelor peak retains
Between be 9.415min.
Figure 15 is the HPLC figures of mixed solution under 2 chromatographic condition of contrast test, and impurity C retention times are 11.224min, miscellaneous
Matter A retention times are 12.519min, and separating degree is preferable, but the chromatographic peak of ticagrelor and impurity B almost overlaps, no
It can accurate checked for impurities B.
Contrast test 3：
Chromatographic column：AD-H, 4.6mm × 250mm, 5 μm；
Mobile phase：N-hexane-ethyl alcohol (85:15)
Solvent：Mobile phase
Column temperature：30℃；Flow velocity：1.0ml/min；UV detectors (Detection wavelength 242nm).
Sampling volume：20μL.
It takes impurity A reference substance appropriate, is dissolved with solvent, be configured to impurity A reference substance solutions of every 1mL containing about 25 μ g.
It takes impurity B reference substance appropriate, is dissolved with solvent, be configured to impurity B reference substance solutions of every 1mL containing about 50 μ g.
It takes impurity C reference substances appropriate, is dissolved with solvent, be configured to impurity C reference substance solutions of every 1mL containing about 5 μ g.
Take impurity A, impurity B, impurity C reference substance solutions appropriate, it is mixed containing each 0.5 μ g of impurity that solubilizer is diluted to every 1ml
Close dirt solution.
It takes ticagrelor sample appropriate, is dissolved with solvent, be configured to test solutions of every 1mL containing about 0.5mg.
Take ticagrelor and impurity A, impurity B, impurity C reference substance solutions appropriate, solubilizer is diluted to every 1ml containing each impurity
0.5 μ g, the mixed solution of ticagrelor 0.5mg.
Measuring method：Above-mentioned 20 μ L of solution injections liquid chromatograph is taken, chromatogram is recorded, as a result as shown in Figure 16~21.
Figure 16 is the HPLC figures of impurity A reference substance solution under 3 chromatographic condition of contrast test, and the retention time of impurity A is
32.182min。
Figure 17 is the HPLC figures of impurity B reference substance solution under 3 chromatographic condition of contrast test, and the retention time of impurity B is
21.799min。
Figure 18 is the HPLC figures of impurity C reference substance solutions under 3 chromatographic condition of contrast test, and the retention time of impurity C is
27.382min。
Figure 19 is the HPLC figures of ticagrelor test solution under 3 chromatographic condition of contrast test, when ticagrelor peak retains
Between be 19.806min.
Figure 20 is the HPLC figures of poly-doped impurity solution under 3 chromatographic condition of contrast test, and impurity B retention time is
22.099min, impurity C retention time are 27.480min, and impurity A retention time is 32.733min.
Figure 21 is that the HPLC of test sample and impurity mixed solution schemes under 3 chromatographic condition of contrast test, and impurity C retention times are
27.392min, impurity A retention time are 32.541min, and separating degree is preferable, but by impurity B at ticagrelor main peak hangover
Chromatographic peak masks.Therefore, the method for contrast test 3 can not accurate checked for impurities B.
Advantageous effect in order to further illustrate the present invention, the present invention provide tests below example.
The methodological study of test example 1, detection method
Various experiments use following condition in this test example：
Chromatographic column：AD-H, 4.6mm × 250mm, 5 μm；
Mobile phase：N-hexane-ethyl alcohol (90:10) 0.1%~0.5% diethylamine, is added in ethyl alcohol
Solvent：Mobile phase
Column temperature：30℃；Flow velocity：1.0ml/min；UV detectors (Detection wavelength 242nm).
Sampling volume：20μL.
1st, Detection wavelength
Ticagrelor is taken, dissolved with solvent and dilutes the solution that suitable concentration is made, according to UV-VIS spectrophotometry
(two annex IVA of Chinese Pharmacopoeia version in 2010) carry out spectral scan, ultraviolet spectrogram such as Figure 22 in the range of 200~400nm
It is shown.
The results show that ticagrelor has absorption in 200~300nm, impurity A, impurity B and impurity C are ticagrelor
Chiral isomer, ultraviolet spectrogram indistinction, therefore the selection in the range of 200~300nm is selected to measure wavelength, and for lattice
Rui Luo is detected at 242nm wavelength, and baseline noise is smaller, and other impurities are noiseless to impurity A, impurity B and impurity C, therefore, most
Select the Detection wavelength that 242nm is detected as ticagrelor chiral isomer eventually.
2nd, specificity is tested
It takes ticagrelor appropriate, after being dissolved with ethyl alcohol, adds flowing phase dilution that the solution in every 1mL containing about 0.5mg is made, make
For test solution.Separately take impurity A in ticagrelor, impurity B, impurity C appropriate, impurity reference substance solution separately takes impurity to compare
Product solution mixes, and poly-doped impurity solution is prepared with flowing phase dilution.Precision takes above-mentioned poly-doped impurity reference substance solution, for examination respectively
Product solution and each 20 μ L of solvent inject liquid chromatograph, record chromatogram.As a result as shown in figs. 1 to 6.
The result shows that under conditions of detection method, remaining impurity pair in ticagrelor sample and ticagrelor
Impurity A, impurity B, the measure of impurity C is noiseless, it was demonstrated that the specificity of detection method is strong.
3rd, standard curve and the range of linearity
Precision measurement impurity A, impurity B, impurity C reference substance solutions are appropriate, and a series of concentration are made with flowing phase dilution
Reference substance solution.The accurate each 20 μ L of reference substance solution for taking various concentration respectively, inject liquid chromatograph, record chromatogram.Point
Peak area is not measured, the results are shown in Table 1.
Table 1, linear relationship
With a concentration of abscissa X of impurity reference substance solution, using its peak area as ordinate Y, standard curve is drawn, is calculated
The equation of linear regression and correlation coefficient r of impurity (1R, 2S) -2- (3,4- difluorophenyl) cyclopropylamine, standard curve such as Figure 25 institutes
Show.
The result shows that in detection method the concentration of impurity A in the range of the μ g/mL of 0.275 μ g/mL~2.204 with
Peak area is in good linear relationship, linear equation：Y=22540.6837X-2199.8432, r=0.9994；Impurity B it is dense
Degree is in the range of the μ g/mL of 0.268 μ g/mL~2.145 with peak area in good linear relationship, linear equation：Y=
25575.0896X-1514.7421 r=0.9996；The concentration of impurity C in the range of the μ g/mL of 0.254 μ g/mL~2.034 with peak
Area is in good linear relationship, linear equation：Y=26287.5744X-1548.7861, r=0.9995, it was demonstrated that present invention side
The method range of linearity is wide, and accuracy is high.
In addition, from calibration curve equation and figure as can be seen that slope is far longer than intercept, standard curve is said close to origin
The assay of bright each impurity is suitable for the one point external standard method of the present invention.
4th, precision test
The poly-doped impurity reference substance solution 3# under test example 1 the 3rd is taken, precision takes 20 μ L, injects high performance liquid chromatograph,
Continuous sample introduction 6 times, detection method according to the invention measures peak area respectively, the results are shown in Table 2.
Table 2, Precision test result
The RSD that impurity A peak area is calculated is：1.45%, the RSD of impurity B peak area are：2.54%, impurity C peaks face
Long-pending RSD is：2.26%, it was demonstrated that detection method precision of the invention is excellent.
5th, quantitative limit
It is appropriate to measure the lower poly-doped impurity reference substance solution 6# of test example the 3rd, adds flowing phase dilution, precision measures 20 μ l,
Liquid chromatograph is injected, detection method according to the invention measures peak area and baseline noise, the results are shown in Table 3.
Table 3, quantitative limit result of the test
| Impurity title | Concentration (μ g/mL) | Quantitative limit (ng) |
| Impurity A | 0.138 | 2.76 |
| Impurity B | 0.134 | 2.68 |
| Impurity C | 0.127 | 2.54 |
The peak height of impurity A, impurity B and impurity C is each about 10 times of baseline noise, based on signal-to-noise ratio S/N=10, obtains impurity
Quantifying for A is limited to 2.76ng, and quantifying for impurity B is limited to 2.68ng, and quantifying for impurity C is limited to 2.54ng, it was demonstrated that the method for the present invention
Detection sensitivity it is high, can fully meet the requirement of impurity quantitative determination.
6th, repetitive test
Precision weighs 6 parts of ticagrelor, respectively about 10mg, puts respectively in 20mL measuring bottles, and solubilizer dissolves and is diluted to scale,
Obtain test solution.Precision measures above-mentioned each 20 μ L of 6 parts of test solutions, and detection method according to the invention is detected, presses
External standard method the results are shown in Table 4 with the content of calculated by peak area impurity A, impurity B and impurity C.
Table 4, repetitive test result
| Sample number into spectrum | 1 | 2 | 3 | 4 | 5 | 6 |
| Impurity A | It does not detect | It does not detect | It does not detect | It does not detect | It does not detect | It does not detect |
| Impurity B | It does not detect | It does not detect | It does not detect | It does not detect | It does not detect | It does not detect |
| Impurity C | 0.019% | 0.018% | 0.016% | 0.019% | 0.017% | 0.020% |
From the above results, the repeatability of detection method is good.
7th, solution stability testing
Precision weighs ticagrelor 101mg, puts in 20mL measuring bottles, and solubilizer dissolves and is diluted to scale, and it is molten to obtain test sample
Liquid.The 20 μ L of 0h, 1h, 2h, 4h, 6h, 8h sample introduction after preparation, record chromatogram, investigate impurity A in its test solution,
The steadiness of impurity B and impurity C, the results are shown in Table 5.
Table 5, test solution stability test result table
| Standing time | 0h | 2h | 4h | 6h | 8h |
| Impurity A | It does not detect | It does not detect | It does not detect | It does not detect | It does not detect |
| Impurity B | It does not detect | It does not detect | It does not detect | It does not detect | It does not detect |
| Impurity C | 0.019% | 0.020% | 0.018% | 0.019% | 0.017% |
From the above results, impurity A, impurity B do not detect in test solution in 8 hours after preparation, impurity C inspections
Go out result without significant change, it was demonstrated that detection method test solution is stablized.
8th, recovery test
Precision weighs 9 parts of ticagrelor, respectively about 10mg, puts respectively in 20mL measuring bottles, adds in each impurity under test example the 3rd
Concentration is about each 3 parts of reference substance solution 0.5mL, 1.0mL, 2.0mL of 20 μ g/mL, and solubilizer dissolves and is diluted to scale, shakes
It is even, respectively as rate of recovery test solution.Accurate pair taken under 9 parts of rate of recovery test solutions and test example the 4th respectively
It is measured according to each 20 μ L sample introductions of product solution, records chromatogram, calculate measured amount, the reference substance addition of impurity A, impurity B and impurity C
And the rate of recovery, it the results are shown in Table 6~8.
Calculation formula：
In formula：A is the amount (μ g) of contained isomer impurities in test sample；
B is isomer impurities reference substance addition (μ g)；
C is the measured amount (μ g) of isomer impurities.
Table 6, impurity A recovery test result
The result shows that detection method measures the chiral isomer impurity A in ticagrelor, the rate of recovery exists
Between 100.6%~106.0%, relative standard deviation 1.75%, it was demonstrated that the detection method rate of recovery of the invention is good, accuracy
It is high.
Table 7, impurity B recovery test result
The result shows that detection method measures the chiral isomer impurity B in ticagrelor, the rate of recovery is 98.9%
Between~103.6%, relative standard deviation 1.90%, it was demonstrated that the detection method rate of recovery of the invention is good, and accuracy is high.
Table 8, impurity C recovery test results
The result shows that detection method measures the chiral isomer impurity C in ticagrelor, the rate of recovery is 98.4%
Between~103.0%, relative standard deviation 1.74%, it was demonstrated that the detection method rate of recovery of the invention is good, and accuracy is high.
In conclusion the present invention provides a kind of new detection method of isomer impurities content in ticagrelor, each chromatography
Separating degree between peak is high, noiseless between each other, can realize simultaneously to impurity B and impurity A, the accurate detection of impurity C, and
And it is easy to operate, it is easy to control, testing cost is low, and with good linear relationship, specificity, precision, stability, spirit
Sensitivity and repeatability, sample recovery rate is high, and testing result is accurate, reliable, for isomer impurities in monitoring ticagrelor drug
Content provides a kind of effective detection method, further ensures the product quality of ticagrelor and the medication peace of patient
Entirely.
Claims (10)
1. a kind of detection method of isomer impurities content in ticagrelor, it is characterised in that：It includes the following steps：
A, the reference substance of isomer impurities is taken, prepares reference substance solution；
B, ticagrelor sample to be checked is taken, prepares test solution；
C, reference substance solution, test solution are detected respectively using efficient liquid-phase chromatography method, the high-efficient liquid phase color
The testing conditions of spectral method are：
Chromatographic column：Stationary phase is amylose-three (3,5- xylyls carbamate)；
Mobile phase：The volume ratio of n-hexane-ethyl alcohol, n-hexane and ethyl alcohol is 88%：12%~92%：8%, in the ethyl alcohol
Diethylamine containing 0.1%~0.5%ml/ml；
Detection wavelength：230nm~250nm；
D, it is obtained auspicious for lattice with calculated by peak area or according to the standard curve calculating between peak area and concentration by external standard method
Isomer impurities content in the sample of Lip river；
The isomer impurities are simultaneously including (1R, 2R, 3S, 5R) -3- [7- { [(1R, 2S) -2- (3,4- difluorophenyls) rings third
Base] amino } -5- rosickyite base -3H-1,2,3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- two
Alcohol, (1R, 2R, 3S, 5R) -3- [7- { [(1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl] amino } -5- rosickyite base -3H-1,2,
3- triazoles [4,5-d] pyrimidin-3-yl] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol and (1S, 2S, 3R, 5S) -3- [7-
{ [(1S, 2R) -2- (3,4- difluorophenyls) cyclopropyl] amino } -5- rosickyite base -3H-1,2,3- triazole [4,5-d] pyrimidines -3-
Base] -5- (2- hydroxy ethoxies) pentamethylene -1,2- glycol.
2. detection method according to claim 1, it is characterised in that：In step a, the solvent for preparing reference substance solution is step
Mobile phase in rapid c；In step b, the solvent for preparing test solution is the mobile phase in step c.
3. detection method according to claim 1, it is characterised in that：In step c, the chromatographic column is CHIRALPAK®
AD-H, length 250mm, internal diameter 4.6mm, the grain size of filler is 5 μm.
4. detection method according to claim 1, it is characterised in that：In step c, the column temperature of the chromatographic column for 25 DEG C~
35℃。
5. detection method according to claim 1, it is characterised in that：In step c, the volume ratio of n-hexane and ethyl alcohol is
90%：10%；Diethylamine containing 0.2%ml/ml in the ethyl alcohol.
6. detection method according to claim 1, it is characterised in that：In step c, the flow velocity of the mobile phase is 0.9ml/
Min~1.1ml/min.
7. detection method according to claim 6, it is characterised in that：The flow velocity of the mobile phase is 1.0ml/min.
8. detection method according to claim 1, it is characterised in that：In step c, the Detection wavelength is 242nm.
9. detection method according to claim 1, it is characterised in that：In step c, sampling volume is the μ l of 10 μ l~50.
10. detection method according to claim 9, it is characterised in that：Sampling volume is 20 μ l.
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