CN105301142A - Method for detecting Ticagrelor and related substances by use of performance liquid chromatography - Google Patents
Method for detecting Ticagrelor and related substances by use of performance liquid chromatography Download PDFInfo
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- CN105301142A CN105301142A CN201510855765.8A CN201510855765A CN105301142A CN 105301142 A CN105301142 A CN 105301142A CN 201510855765 A CN201510855765 A CN 201510855765A CN 105301142 A CN105301142 A CN 105301142A
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- ticagrelor
- buffered saline
- intermediate product
- saline solution
- acetonitrile
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- OEKWJQXRCDYSHL-FNOIDJSQSA-N ticagrelor Chemical compound C1([C@@H]2C[C@H]2NC=2N=C(N=C3N([C@H]4[C@@H]([C@H](O)[C@@H](OCCO)C4)O)N=NC3=2)SCCC)=CC=C(F)C(F)=C1 OEKWJQXRCDYSHL-FNOIDJSQSA-N 0.000 title claims abstract description 70
- 229960002528 ticagrelor Drugs 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000000126 substance Substances 0.000 title claims abstract description 29
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 71
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 19
- 239000007975 buffered saline Substances 0.000 claims abstract description 19
- 238000010828 elution Methods 0.000 claims abstract description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 4
- 238000001514 detection method Methods 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 11
- 239000012895 dilution Substances 0.000 claims description 11
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 9
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 9
- 235000019800 disodium phosphate Nutrition 0.000 claims description 9
- 239000000337 buffer salt Substances 0.000 claims description 8
- 238000004090 dissolution Methods 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 3
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 239000012535 impurity Substances 0.000 abstract description 27
- 238000004458 analytical method Methods 0.000 abstract description 13
- 239000000945 filler Substances 0.000 abstract 1
- 239000013067 intermediate product Substances 0.000 description 64
- 230000014759 maintenance of location Effects 0.000 description 37
- 239000012071 phase Substances 0.000 description 17
- 239000000243 solution Substances 0.000 description 10
- 238000000926 separation method Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 239000007791 liquid phase Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000005259 measurement Methods 0.000 description 6
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 229910052698 phosphorus Inorganic materials 0.000 description 5
- 239000011574 phosphorus Substances 0.000 description 5
- 239000012749 thinning agent Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229940086777 brilinta Drugs 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for detecting Ticagrelor and related substances by use of performance liquid chromatography. The chromatographic conditions of the method are as follows: octadecylsilane chemically bonded silica is taken as a chromatographic column filling agent, a buffered saline solution and acetonitrile are taken as mobile phases for gradient elution, and an ultraviolet detector is taken as a detector. The method provided by the invention can well separate Ticagrelor from impurities, is smooth in baseline, short in analysis time and high in efficiency, and is simple and reliable in detecting Ticagrelor-related substances.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, be specifically related to a kind of method adopting high performance liquid chromatography to detect ticagrelor and related substance thereof.
Background technology
Ticagrelor, Ticagrelor, its chemical name is (1S, 2S, 3R, 5S)-3-(7-(1R, 2S)-2-(3,4-difluorophenyl) cyclopropylamino)-5-third sulfydryl-3H-[1,2,3] triazole-[4,5-d] pyrimidin-3-yl)-5-(2-hydroxy ethoxy) cyclopentane-1,2-glycol.
Ticagrelor structural formula is:
Ticagrelor is RA233, for the treatment of acute coronary artery syndrome (ACS).Ticagrelor sheet is developed by Britain's AstraZeneca (AstraZeneca) drugmaker, and obtain U.S. FDA approval listing in July, 2011, commodity are called Brilinta.
The difficulty that ticagrelor and related substance thereof detect is, the impurity that product produces in building-up process is more, and issuable impurity comprises the plurality of impurities such as crucial starting material, residual intermediate product, and the impurity of ticagrelor is not easily separated completely.At present, the related substance detection method of ticagrelor is reported without pertinent literature.
Summary of the invention
The object of this invention is to provide a kind of method adopting high performance liquid chromatography to detect ticagrelor and related substance thereof.Ticagrelor and related substance (i.e. impurity) thereof can effectively be separated by the method, and analysis time is short, thus achieves effective detection of ticagrelor and impurity thereof, ensure that the quality control of ticagrelor.
Specifically, a kind of method adopting high performance liquid chromatography to detect ticagrelor and related substance thereof of the present invention, comprise: the chromatographic column taking octadecylsilane chemically bonded silica as Stationary liquid, with buffered saline solution and acetonitrile as mobile phase, take UV-detector as detecting device, adopt linear gradient elution method to measure.
The method of the invention described above, chromatographic column, be preferably C18 post, specification is 4.6 × 150mm, 1.8 μm, wherein, the temperature of chromatographic column is 20 ~ 30 DEG C, optimum column temperature is 25 DEG C, described flow rate of mobile phase is 0.6 ~ 1.2ml/min, preferred 0.8ml/min, the determined wavelength of described UV-detector is 230 ~ 250nm, preferred detection wavelength is 242nm, the pH scope of described buffered saline solution is 5.0 ~ 6.0, wherein, buffer salt is selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate, one or more in sodium dihydrogen phosphate, be preferably the aqueous solution of sodium hydrogen phosphate, be 5.0 ~ 6.0 by phosphoric acid adjust ph.
The method of the invention described above, described linear gradient elution method, wherein, the gradient of buffered saline solution and acetonitrile volume ratio is as following table 1:
The gradient of table 1 buffered saline solution and acetonitrile volume ratio
| Time (minute) | Buffered saline solution ratio (%) | Acetonitrile ratio (%) |
| 0 | 5％～95％ | 95％～5％ |
| 5 | 5％～95％ | 95％～5％ |
| 25 | 5％～95％ | 95％～5％ |
| 45 | 5％～95％ | 95％～5％ |
| 55 | 5％～95％ | 95％～5％ |
| 60 | 5％～95％ | 95％～5％ |
Preferably, method of the present invention, described linear gradient elution method, wherein, the gradient of buffered saline solution and acetonitrile volume ratio is as following table 2:
The gradient of table 2 buffered saline solution and acetonitrile volume ratio
| Time (minute) | Buffered saline solution ratio (%) | Acetonitrile ratio (%) |
| 0 | 70％～80％ | 30％～20％ |
| 5 | 70％～80％ | 30％～20％ |
| 25 | 55％～65％ | 45％～35％ |
| 45 | 35％～45％ | 65％～55％ |
| 55 | 15％～25％ | 85％～75％ |
| 60 | 15％～25％ | 85％～75％ |
The method of the invention described above, specific operation process comprises gets ticagrelor in right amount, and weigh, use dilution ultrasonic dissolution, be made into the sample solution of every 1ml containing ticagrelor 1mg, flow velocity is 0.8ml/ml, and determined wavelength is 242nm, column temperature 25 DEG C; Precision measures sample solution 10 μ l injection liquid chromatography, and complete ticagrelor and related substance detection, described thinning agent is acetonitrile: the mixed solvent of water=35:65v/v.
In one embodiment, a kind of method adopting high performance liquid chromatography to detect ticagrelor and related substance thereof of the present invention, comprise: the chromatographic column taking octadecylsilane chemically bonded silica as Stationary liquid, described chromatographic column is preferably C18 post, specification is 4.6 × 150mm, 1.8 μm, wherein, the temperature of chromatographic column is 20 ~ 30 DEG C, be preferably 25 DEG C, with buffered saline solution and acetonitrile as mobile phase, the pH scope of described buffered saline solution is 5.0 ~ 6.0, be preferably 5.0 ~ 6.0 by phosphoric acid adjust ph, described buffer salt is selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate, one or more in sodium dihydrogen phosphate, be preferably the aqueous solution of sodium hydrogen phosphate, take UV-detector as detecting device, the determined wavelength of described UV-detector is 230 ~ 250nm, preferred detection wavelength is 242nm, employing linear gradient elution method measures, described linear gradient elution method, wherein, the gradient as above table 1 of buffered saline solution and acetonitrile volume ratio, preferred as above table 2.
A kind of high performance liquid chromatography of the present invention detects the method for ticagrelor and related substance thereof, comprises following operation:
Get this product (i.e. ticagrelor) appropriate, weigh, with dilution (acetonitrile-water=35:65v/v) ultrasonic dissolution, be made into the sample solution of every 1ml containing ticagrelor 1mg, flow velocity is 0.8ml/ml, and determined wavelength is 240nm, column temperature 25 DEG C; Precision measures sample solution 10 μ l injection liquid chromatography, completes the detection of ticagrelor and related substance thereof.
The advantage of method of the present invention is: between each impurity, degree of separation is good, and each impurity is separated with ticagrelor thoroughly, and simple and efficient to handle, analysis time is short, and cost is low, effectively can control the related substance of ticagrelor, guarantees security and the validity of product.
In embodiments of the invention, described number percent (%) all refers to percent by volume except indicating especially.
In embodiments of the invention, described related substance includes but not limited to the impurity of ticagrelor: intermediate product A, intermediate product B, intermediate product C, SM2, SM3;
The chemical structural formula of intermediate product A is as follows:
The chemical structural formula of intermediate product B is as follows:
The chemical structural formula of intermediate product C is as follows:
The chemical structural formula of SM2 is as follows:
The chemical structural formula of SM3 is as follows:
Accompanying drawing explanation
Fig. 1 is the chromatogram of embodiment 1.
Fig. 2 is the chromatogram of embodiment 2.
Fig. 3 is the chromatogram of embodiment 3.
Fig. 4 is the chromatogram of comparative example.
Embodiment:
Following examples for illustration of the present invention, but not as limiting the scope of the invention.
Embodiment 1
Instrument and condition
Agilent 1200 high performance liquid chromatograph, C18 post (4.6 × 150mm, 1.8 μm), be mobile phase A with buffer salt solution (the sodium hydrogen phosphate aqueous solution of 10mmol/L, with phosphorus acid for adjusting pH to 5.0), using acetonitrile as Mobile phase B, determined wavelength is 242nm, flow velocity 0.8ml/min, column temperature 25 DEG C, dilution is acetonitrile: water (35:65v/v).Sample size is 10 μ l.Wash-out is carried out by such as following table 3 Gradient program:
The gradient of table 3 mobile phase A and B
Experimental procedure:
Get ticagrelor and impurity such as intermediate product A, intermediate product B, intermediate product C, SM2, SM3 are made into 1ml solution and thinning agent separately, high-efficient liquid phase analysis is carried out respectively by above-mentioned condition, record chromatogram, obtain the location retention time of ticagrelor and each impurity, the retention time (min) of ticagrelor, intermediate product A, intermediate product B, intermediate product C, SM2, SM3 is respectively 33.05,34.46,29.36,50.73,32.23,20.33, and thinning agent is noiseless.
Get this product (i.e. ticagrelor) appropriate, weigh, use dilution ultrasonic dissolution, and add the impurity (intermediate product A, intermediate product B, intermediate product C, SM2, SM3) of ticagrelor, be made into the mixed solution of every 1ml containing ticagrelor, intermediate product A, intermediate product B, each 20 μ g of intermediate product C, SM2, SM3.High-efficient liquid phase analysis is carried out, record chromatogram by above-mentioned condition.The results are shown in Figure 1 and table 4.
Table 4 measurement result
| Peak # | Retention time | Area | Degree of separation | Theoretical tray | Highly (mAu) | Symmetrical factor |
| 1 | 20.343 | 3223.83 | —— | 210193 | 454.50 | 0.81 |
| 2 | 29.370 | 2581.21 | 41.54 | 209938 | 259.16 | 0.86 |
| 3 | 32.244 | 1746.17 | 12.12 | 353382 | 203.98 | 0.85 |
| 4 | 33.062 | 2922.51 | 3.82 | 394521 | 350.66 | 0.91 |
| 5 | 34.468 | 2634.06 | 6.66 | 426563 | 316.09 | 0.90 |
| 6 | 50.728 | 2306.72 | 76.16 | 888962 | 273.08 | 0.79 |
In conjunction with the location retention time of ticagrelor and each impurity, the chromatographic peak of Fig. 1 retention time to be the chromatographic peak of 20.343min be SM3, the chromatographic peak of retention time to be the chromatographic peak of 29.370min be intermediate product B, the chromatographic peak of retention time to be the chromatographic peak of 32.244min be SM2, the chromatographic peak of retention time to be the chromatographic peak of 33.062min be ticagrelor, the chromatographic peak of retention time to be the chromatographic peak of 34.468min be intermediate product A, the chromatographic peak of retention time to be the chromatographic peak of 50.728min be intermediate product C.Under above-mentioned chromatographic condition, between the impurity of ticagrelor, degree of separation is good, and terminates the detection of whole related substance in 60min, and analysis time is short, and efficiency is high, cost-saving, meets the requirement of Chinese Pharmacopoeia.
Embodiment 2
Instrument and condition
Agilent 1200 high performance liquid chromatograph, C18 post (4.6 × 150mm, 1.8 μm), be mobile phase A with buffer salt solution (the sodium hydrogen phosphate aqueous solution of 10mmol/L, with phosphorus acid for adjusting pH to 6.0), using acetonitrile as Mobile phase B, determined wavelength is 242nm, flow velocity 0.8ml/min, column temperature 25 DEG C, dilution is acetonitrile: water (35:65V/V).Sample size is 10 μ l.Wash-out is carried out by the Gradient program of such as following table 5:
The gradient of table 5 mobile phase A and B
Experimental procedure:
Get ticagrelor and impurity is made into 1ml solution separately as intermediate product A, intermediate product B, intermediate product C, SM2, SM3, carry out high-efficient liquid phase analysis respectively by above-mentioned condition, record chromatogram, obtains the location retention time of ticagrelor and each impurity.The retention time (min) of ticagrelor, intermediate product A, intermediate product B, intermediate product C, SM2, SM3 is respectively 36.66,37.40,33.20,53.15,35.87,24.57, and thinning agent is noiseless.
Get this product (i.e. ticagrelor) appropriate, weigh, use dilution ultrasonic dissolution, and add the impurity (intermediate product A, intermediate product B, intermediate product C, SM2, SM3) of ticagrelor, be made into the mixed solution of every 1ml containing ticagrelor, intermediate product A, intermediate product B, each 20 μ g of intermediate product C, SM2, SM3.High-efficient liquid phase analysis is carried out, record chromatogram by above-mentioned condition.The results are shown in Figure 2 and table 6.
Table 6 measurement result
| Peak # | Retention time | Area | Degree of separation | Theoretical tray | Highly (mAu) | Symmetrical factor |
| 1 | 24.571 | 3258.78 | —— | 296086 | 457.98 | 0.86 |
| 2 | 33.208 | 2431.28 | 40.97 | 305258 | 262.52 | 0.90 |
| 3 | 35.885 | 1682.74 | 11.80 | 455436 | 203.55 | 0.90 |
| 4 | 36.650 | 2881.15 | 3.63 | 497952 | 355.60 | 0.97 |
| 5 | 37.995 | 2563.45 | 6.40 | 514074 | 311.08 | 0.96 |
| 6 | 53.163 | 2301.38 | 75.08 | 1234145 | 309.86 | 0.84 |
In conjunction with the location retention time of ticagrelor and each impurity, the chromatographic peak of retention time to be the chromatographic peak of 24.571min be SM3 in Fig. 2, the chromatographic peak of retention time to be the chromatographic peak of 33.208min be intermediate product B, the chromatographic peak of retention time to be the chromatographic peak of 35.885min be SM2, the chromatographic peak of retention time to be the chromatographic peak of 36.650min be ticagrelor, the chromatographic peak of retention time to be the chromatographic peak of 37.995min be intermediate product A, the chromatographic peak of retention time to be the chromatographic peak of 53.163min be intermediate product C.Under above-mentioned chromatographic condition, between the impurity of ticagrelor, degree of separation is good, and terminates the detection of whole related substance in 60min, and analysis time is short, and efficiency is high, cost-saving, meets the requirement of Chinese Pharmacopoeia.
Embodiment 3
Instrument and condition
Agilent 1200 high performance liquid chromatograph, C18 post (4.6 × 150mm, 1.8 μm), be mobile phase A with buffer salt solution (the sodium hydrogen phosphate aqueous solution of 10mmol/L, with phosphorus acid for adjusting pH to 6.0), using acetonitrile as Mobile phase B, determined wavelength is 242nm, flow velocity 0.8ml/min, column temperature 25 DEG C, dilution is acetonitrile: water (35:65V/V).Sample size is 10 μ l.Wash-out is carried out by such as following table 7 Gradient program:
The gradient of table 7 mobile phase A and B
Experimental procedure:
Get ticagrelor and impurity is made into 1ml solution separately as intermediate product A, intermediate product B, intermediate product C, SM2, SM3, carry out high-efficient liquid phase analysis respectively by above-mentioned condition, record chromatogram, obtains the location retention time of ticagrelor and each impurity.The retention time (min) of ticagrelor, intermediate product A, intermediate product B, intermediate product C, SM2, SM3 is respectively 39.72,44.01,36.51,55.09,39.00,28.55, and thinning agent is noiseless.
Get this product (i.e. ticagrelor) appropriate, weigh, use dilution ultrasonic dissolution, and add the impurity (intermediate product A, intermediate product B, intermediate product C, SM2, SM3) of ticagrelor, be made into the mixed solution of every 1ml containing ticagrelor, intermediate product A, intermediate product B, each 20 μ g of intermediate product C, SM2, SM3.High-efficient liquid phase analysis is carried out, record chromatogram by above-mentioned condition.The results are shown in Figure 3 and table 8 measurement result.
Table 8 measurement result
| Peak # | Retention time | Area | Degree of separation | Theoretical tray | Highly (mAu) | Symmetrical factor |
| 1 | 28.566 | 3287.51 | —— | 431729 | 471.96 | 0.77 |
| 2 | 36.506 | 2597.88 | 39.53 | 412967 | 292.53 | 0.82 |
| 3 | 39.002 | 1762.63 | 11.50 | 572389 | 215.27 | 0.80 |
| 4 | 39.727 | 2933.46 | 3.57 | 635025 | 368.85 | 0.85 |
| 5 | 41.027 | 2635.85 | 6.45 | 652300 | 325.58 | 0.85 |
| 6 | 55.083 | 2328.00 | 72.38 | 424807 | 319.33 | 0.74 |
In conjunction with the location retention time of ticagrelor and each impurity, the chromatographic peak of retention time to be the chromatographic peak of 28.566min be SM3 in Fig. 3, the chromatographic peak of retention time to be the chromatographic peak of 36.506min be intermediate product B, the chromatographic peak of retention time to be the chromatographic peak of 39.002min be SM2, the chromatographic peak of retention time to be the chromatographic peak of 39.727min be ticagrelor, the chromatographic peak of retention time to be the chromatographic peak of 41.027min be intermediate product A, the chromatographic peak of retention time to be the chromatographic peak of 55.083min be intermediate product C.Under above-mentioned chromatographic condition, between the impurity of ticagrelor, degree of separation is good, and terminates the detection of whole related substance in 60min, and analysis time is short, and efficiency is high, cost-saving, meets the requirement of Chinese Pharmacopoeia.
Comparative example
Ticagrelor sheet import quality standard (import standard No.: JX20110193) discloses ticagrelor sheet related substance detection method, we adopt the method to detect ticagrelor related substance (impurity: intermediate product A, intermediate product B, intermediate product C, SM2, SM3), specific as follows:
Instrument and condition
Agilent 1200 high performance liquid chromatograph, C18 post (4.6 × 150mm, 1.8 μm), [biphosphate sodium water solution (phosphorus acid for adjusting pH to the 3.0) 10ml of 1.0mol/L is got with buffer salt solution, add water to 900ml, shaking up]-acetonitrile (90:10) is mobile phase A, [biphosphate sodium water solution (phosphorus acid for adjusting pH to the 3.0) 10ml of 1.0mol/L is got with buffer salt solution, add water to 300ml, shaking up]-acetonitrile (30:70) is Mobile phase B, determined wavelength is 242nm, flow velocity 1.0ml/min, column temperature 55 DEG C, dilution is acetonitrile: water (35:65v/v).Sample size is 10 μ l.Wash-out is carried out by such as following table 9 Gradient program:
The gradient of table 9 mobile phase A and B
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 7 | 35 | 65 |
| 15 | 35 | 65 |
| 16 | 0 | 100 |
| 23 | 0 | 100 |
| 25 | 90 | 10 |
| 30 | 90 | 10 |
Experimental procedure:
Get this product (i.e. ticagrelor) appropriate, weigh, use dilution ultrasonic dissolution, and add the impurity (intermediate product A, intermediate product B, intermediate product C, SM2, SM3) of ticagrelor, be made into the mixed solution of every 1ml containing ticagrelor, intermediate product A, intermediate product B, each 20 μ g of intermediate product C, SM2, SM3.High-efficient liquid phase analysis is carried out, record chromatogram by above-mentioned condition.The results are shown in Figure 4 and table 10 measurement result.
Table 10 measurement result
| Peak # | Retention time | Area | Degree of separation | Theoretical tray | Highly (mAu) | Symmetrical factor |
| 1 | 6.120 | 2622.29 | —— | 93631 | 914.32 | 0.88 |
| 2 | 7.316 | 2066.49 | 14.72 | 126923 | 707.74 | 0.91 |
| 3 | 7.605 | 1369.29 | 3.58 | 147794 | 489.36 | 1.00 |
| 4 | 7.687 | 2446.89 | 1.02 | 139262 | 842.23 | 0.92 |
| 5 | 7.895 | 2078.76 | 2.52 | 146170 | 720.74 | 0.90 |
| 6 | 9.986 | 1943.58 | 24.32 | 201163 | 596.02 | 0.86 |
The chromatographic peak of retention time to be the chromatographic peak of 6.120min be SM3 in Fig. 4, the chromatographic peak of retention time to be the chromatographic peak of 7.316min be intermediate product B, the chromatographic peak of retention time to be the chromatographic peak of 7.605min be SM2, the chromatographic peak of retention time to be the chromatographic peak of 7.687min be ticagrelor, the chromatographic peak of retention time to be the chromatographic peak of 7.895min be intermediate product A, the chromatographic peak of retention time to be the chromatographic peak of 9.986min be intermediate product C.Under above-mentioned chromatographic condition, ticagrelor peak fails effectively to be separated with adjacent SM2 peak, and degree of separation cannot meet the requirement of Chinese Pharmacopoeia.
Claims (10)
1. the method for a high performance liquid chromatography detection ticagrelor and related substance thereof, comprising with octadecylsilane chemically bonded silica is the chromatographic column of Stationary liquid, with buffered saline solution and acetonitrile as mobile phase, take UV-detector as detecting device, adopt linear gradient elution method to measure.
2. method according to claim 1, wherein, described flow rate of mobile phase is 0.6 ~ 1.2ml/min, preferred 0.8ml/min.
3. method according to claim 1, wherein, the determined wavelength of described UV-detector is 230 ~ 250nm, and preferred detection wavelength is 242nm.
4. method according to claim 1, wherein, the temperature of chromatographic column is 20 ~ 30 DEG C, and optimum column temperature is 25 DEG C.
5. method according to claim 1, described linear gradient elution method, wherein, the gradient of buffered saline solution and acetonitrile volume ratio is:
6. method according to claim 5, described linear gradient elution method, wherein, the gradient of buffered saline solution and acetonitrile volume ratio is:
7., according to the arbitrary described method of claim 1-6, described buffer salt is selected from one or more in dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate.
8., according to the arbitrary described method of claim 1-6, wherein, the pH scope of buffered saline solution is 5.0 ~ 6.0.
9. method according to claim 7, wherein, described buffered saline solution is the aqueous solution of sodium hydrogen phosphate, is 5.0 ~ 6.0 by phosphoric acid adjust ph.
10. method according to claim 1, comprises and gets ticagrelor in right amount, weigh, use dilution ultrasonic dissolution, and be made into the sample solution of every 1ml containing ticagrelor 1mg, flow velocity is 0.8ml/ml, and determined wavelength is 242nm, column temperature 25 DEG C; Precision measures sample solution 10 μ l injection liquid chromatography, completes active component and related substance in ticagrelor and detects.
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