CN104316637A - Method for determining apixaban cleaning residues by high performance liquid chromatography - Google Patents
Method for determining apixaban cleaning residues by high performance liquid chromatography Download PDFInfo
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- CN104316637A CN104316637A CN201410596393.7A CN201410596393A CN104316637A CN 104316637 A CN104316637 A CN 104316637A CN 201410596393 A CN201410596393 A CN 201410596393A CN 104316637 A CN104316637 A CN 104316637A
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Abstract
The invention relates to a method for determining apixaban cleaning residues by high performance liquid chromatography, and belongs to the field of chemical pharmaceutical detection. The invention aims at providing a method for determining the apixaban cleaning residues. The method is easy and quick to operate and accurate and reliable in determination results to evaluate a cleaning effect so as to ensure medicine quality and medication safety. The high performance liquid chromatography for determining the apixaban cleaning residues, which is disclosed by the invention, is easy and quick to operate, and accurate and reliable in the determination results, so that the cleaning effect of the equipment can be evaluated effectively. According to the high performance liquid chromatography, a 280 nm flow phase is selected, so that the detection limit is reduced effectively. A chromatographic column temperature selects 40 DEG C, so that the peak type can be improved effectively, and minimal residues can be effectively detected; methanol is selected to serve as a solvent, and is not absorbed in the wavelength, so that the interference on the detection caused by the solvent is avoided, and the reliability of the detection method is improved.
Description
Technical field
The present invention relates to a kind of high effective liquid chromatography for measuring Eliquis cleaning residual quantity, belong to chemistry
Pharmacy detection field.
Background technology
The problem of total equipment is inevitably related in production of raw medicine technique, product is switched in total link, if equipment cleaning not exclusively, the last material produced remains in the material will inevitably brought into and produce next time, due to the difference of drug toxicity, the size of residual quantity can directly have influence on Drug safety and drug quality, therefore to control the residual reasonable limit that reaches of cleaning be unusual necessity, and is also the GMP laws and regulations requirement as minimum pharmaceutical requirements.General, allow impurity to exist in the scope that safety allows in product, therefore, adopting the cleaning of the Eliquis in certain instrument analytical method testing product to remain evaluation cleaning performance and drug safety is very necessary, is also very urgent.
At present, the domestic detection method not yet having pharmacopeia and pertinent literature to propose this material, to the more uncorrelated report of the detection of minimal residue.
For Eliquis residual quantity detection, chemistry or general ultraviolet equipment is relied on to be difficult to reach micro-ppm rank testing requirement, also to ensure the good range of linearity simultaneously, good stability, good accuracy and precision, the method for this research can be simple to operate, quick, and measurement result is accurately and reliably, thus effectively can control the residual quantity of Eliquis, ensure medicinal safety and reliability.
Therefore, this research adopts HPLC method, establishes the assay method of a kind of Eliquis cleaning residual quantity.
Summary of the invention
The object of this invention is to provide a kind of simple to operate, quick, the assay method that measurement result Eliquis cleaning is accurately and reliably residual, evaluate cleaning effect, to ensure drug quality and drug safety.
The technical solution used in the present invention is: high effective liquid chromatography for measuring Eliquis cleans, and its innovative point is: comprise sample preparation, sample determination and cubage step; Described sample preparation step is as follows:
(1) blank solution preparation: solvent for cleaning methyl alcohol;
(2) contrast solution stock solution (0.5mg/mL) preparation: take Eliquis reference substance 25mg in 50mL measuring bottle, dissolves with thinning agent, is settled to scale, filters to obtain contrast solution;
(3) contrast solution (0.0005mg/mL) preparation: precision measures contrast stock solution 1.0mL in 100mL measuring bottle, is settled to scale, shakes up; Precision measures above-mentioned solution 1mL in 10mL measuring bottle again, is settled to scale, shakes up;
Described sample determination step is the chromatographic column of Shimadzu Inertsil ODS-3V 5 μm of 250 × 4.6mm for selecting model, adopt double pump system, mobile phase A used: 30mmol/L ammonium acetate solution (takes 2.31g ammonium acetate in 1L water, mixing) B: acetonitrile, setting chromatogram determined wavelength is 280nm, and flow velocity is 1.0 mLmin
-1; Sensitivity is 1.0 AUFS; Column temperature is 40 DEG C; Sample size is 10 μ L, respectively sample solution, contrast solution and blank solution is carried out sample determination;
Described assay step is with the residual quantity of external standard method calculation sample Eliquis.
Further, described mobile phase A is 30mmol/L ammonium acetate solution (taking 2.31g ammonium acetate in 1L water, mixing), and water is 2 redistilled waters, meets HPLC requirement.
Further, Mobile phase B acetonitrile solution is trifluoroacetic acid aqueous solution.
Further, described wavelength is 280nm, changes the maximum absorption wavelength that wavelength is Eliquis, and noiseless to solvent methanol etc.
Further, detecting column temperature is 40 DEG C, effectively can improve peak type and effectively detect minimal residue.
Beneficial effect: this high effective liquid chromatography for measuring Eliquis cleaning residual quantity of the present invention, simple to operate, quick, measurement result accurately and reliably, thus can valuator device cleaning performance effectively; The present invention have employed 280nm in the selection of mobile phase, effectively reduces detection limit; The present invention, on chromatogram column temperature is selected, uses 40 DEG C, effectively can improve peak type and effectively detect minimal residue, methyl alcohol is selected in the selection of solvent, and this wavelength of methyl alcohol, without absorption, is avoided solvent to the interference detected, improved the reliability of detection method.
Accompanying drawing explanation
Be further described below in conjunction with drawings and Examples.
Fig. 1 is specificity of the present invention test collection of illustrative plates.
Fig. 2 is precision test collection of illustrative plates.
Fig. 3 is detectability collection of illustrative plates.
Fig. 4 to be contrast solution be 0.00005 collection of illustrative plates.
Fig. 5 is the collection of illustrative plates of the contrast solution of 1000%.
Fig. 6 is the collection of illustrative plates of the contrast solution of 100%.
Fig. 7 is the collection of illustrative plates of the contrast solution of 150%.
Fig. 8 is the collection of illustrative plates of the contrast solution of 120%.
Fig. 9 is the collection of illustrative plates of the contrast solution of 80%.
Embodiment
Implementation column below can make those skilled in the art more fully understand the present invention, but does not therefore limit the present invention among described scope of embodiments.
embodiment 1
The residual method of high effective liquid chromatography for measuring Eliquis cleaning:
Instrument and reagent: LC-2010A high performance liquid chromatograph (quaternary pump, degas module, UV detecting device, post case, automatic sampler, system monitor, LCsolution) (Japanese Shimadzu Corporation); (other reagent analysis is pure for manufacturer: Jiangsu Baozong Baoda Pharmaceutical Co., Ltd., acetonitrile, methyl alcohol (merck chromatographically pure), and water is the fresh redistilled water of self-control for test sample.
Chromatography condition: chromatographic column: Inertsil ODS-3V 5 μm of 250 × 4.6mm; Mobile phase A: 30mmol/L ammonium acetate solution, Mobile phase B: acetonitrile, isocratic elution; Determined wavelength: 280nm; Computing method: external standard method; Flow velocity: 1.0 mLmin
-1; Sensitivity: 1.0 AUFS; Sample size: 10 μ L; Column temperature 40 DEG C.
Specificity is tested: sample introduction equipment drip washing methyl alcohol, contrast solution sample methyl alcohol and contrast solution respectively, and the specificity of investigation method confirms that this verification method blank does not exist interference to the mensuration of Eliquis; Every day, sample introduction contrast solution, confirmed that the retention time of Eliquis and system suitability meet acceptable standard.Noiseless in blank solution; In contrast solution, the signal to noise ratio (S/N ratio) of main peak is not less than 30.
Solution preparation:
Contrast solution stock solution (0.5mg/mL): get Eliquis reference substance 25mg in 50mL measuring bottle, dissolves with thinning agent, is settled to scale, shakes up.
Contrast solution (0.0005mg/mL): precision measures contrast stock solution 1.0mL in 100mL measuring bottle, is settled to scale, shakes up; Precision measures above-mentioned solution 1mL in 10mL measuring bottle again, is settled to scale, shakes up.
Blank solution: solvent for cleaning
Need testing solution: get whole washing lotion and filter rear direct injected
Sample determination:
1) after system stability, a pin blank solvent is entered: equipment drip washing methyl alcohol
2) a pin contrast solution is entered
3) a pin need testing solution is entered
embodiment 2
The precision test of the inventive method:
System precision: get contrast solution, continuous sample introduction 6 times, calculates the retention time of Eliquis and the RSD of peak area.The actual RSD recording retention time is 0.03%; The RSD of peak area is 0.59%
Method precision: repeat preparation 6 parts of contrast solutions, every part of solution sample introduction 1 time, the RSD of the peak area/sample weighting amount of Eliquis is 0.68%.
Intermediate precision: by another analyst, in another sky, repetition methods precision test on another instrument, the RSD of the peak area/sample weighting amount of Eliquis is 6.87%.
Show that reappearance is good, method is reliable.
embodiment 3
The detectability test of the inventive method:
By the Eliquis reference substance solution of sample introduction low concentration, when signal to noise ratio (S/N ratio) (S/N) is 3, this concentration is its detectability, detectability solution sample introduction 3 pin, calculates and reports the RSD of peak area.Finally determining concentration 0.0125ug/ml when S/N is about 2.8, is detectability.Area RSD is 4.6%.
embodiment 4
The quantitative limit test of the inventive method:
By the Eliquis contrast solution of sample introduction low concentration, when signal to noise ratio (S/N ratio) is 10, this concentration is its quantitative limit, quantitative limit solution sample introduction 6 pin, calculates and reports the RSD of peak area.When finally determining that S/N is about 9.4,0.05ug/ml is quantitative limit, and area RSD is 3.3%.
embodiment 5
The accuracy test of the inventive method.
By simulating actual cleaning condition, detect the concentration of Eliquis in leacheate by the method for inspection of empirical tests.Can select quantitative limit, 50%, 100%, 150% 4 concentration, each concentration repeats 3 times, and the recovery of checking sampling method and repeatability, to verify that the sampling method of leacheate can meet the requirements.
Eliquis stock solution: take Eliquis reference substance 25mg in 50mL measuring bottle, dissolve with thinning agent, constant volume, shakes up.
0.005mg/ml Eliquis solution: precision measures contrast stock solution 1.0mL in 100mL measuring bottle, is settled to scale, shakes up.
50% concentration:
Prepare the glass round bottom flask of a clean 50mL, precision measures the Eliquis methanol solution of the 0.005mg/mL of 1.5mL, as far as possible the uniform inwall drenched at round-bottomed flask, allows round-bottomed flask natural drying.In round-bottomed flask, add the methyl alcohol of 30mL, and be warming up to backflow, be incubated at least 1 hour.Be cooled to and stop backflow, directly get leacheate.Repeat above operation 3 times, get 3 parts of leacheates.
100% concentration:
Prepare the glass round bottom flask of a clean 50mL, precision measures the Eliquis methanol solution of the 0.005mg/mL of 3.0mL, as far as possible the uniform inwall drenched at round-bottomed flask, allows round-bottomed flask natural drying.In round-bottomed flask, add the methyl alcohol of 30mL, and be warming up to backflow, be incubated at least 1 hour.Be cooled to and stop backflow, directly get leacheate.Repeat above operation 3 times, get 3 parts of leacheates.
150% concentration:
Prepare the glass round bottom flask of a clean 50mL, precision measures the Eliquis methanol solution of the 0.005mg/mL of 4.5mL, as far as possible the uniform inwall drenched at round-bottomed flask, allows round-bottomed flask natural drying.In round-bottomed flask, add the methyl alcohol of 30mL, and be warming up to backflow, be incubated at least 1 hour.Be cooled to and stop backflow, directly get leacheate.Repeat above operation 3 times, get 3 parts of leacheates.
Quantitative limit: under measuring item according to quantitative limit, determine the concentration of solution.
Detect the concentration of Eliquis in leacheate by the method for inspection of empirical tests, after system stability, sample introduction blank solution, Eliquis standard solution, leacheate solution, calculate the concentration of 12 samples, and calculate the recovery respectively.
The recovery of the actual recovered rate of quantitative limit level to be the recovery of 88.9,50% level be 90.8%, 100% level is the recovery of 92.3%, 150% level is 90.2%, and average recovery rate is 90.6%
embodiment 6
The solution stability testing of the inventive method
By analyzing the contrast solution placing different time (at least 48h), prove the stability of Eliquis solution.Following time point need be investigated: 0,3h, 6h, 24h and 48h, if find that solution is unstable, need shorten the time interval.Solution appearance should be clarified; Be≤0.89% with the maximum deviation of contrast solution main peak area during 0h, be namely considered as solution-stabilized.
Claims (5)
1. the cleaning of high effective liquid chromatography for measuring Eliquis is residual, it is characterized in that: comprise sample preparation, sample determination and cubage step; Described sample preparation step is as follows:
(1) blank solution preparation: solvent for cleaning methyl alcohol;
(2) contrast solution stock solution (0.5mg/mL) preparation: take Eliquis reference substance 25mg in 50mL measuring bottle, dissolves with thinning agent, is settled to scale, filters to obtain contrast solution;
(3) contrast solution (0.0005mg/mL) preparation: precision measures contrast stock solution 1.0mL in 100mL measuring bottle, is settled to scale, shakes up; Precision measures above-mentioned solution 1mL in 10mL measuring bottle again, is settled to scale, shakes up;
Described sample determination step is the chromatographic column of Shimadzu Inertsil ODS-3V 5 μm of 250 × 4.6mm for selecting model, adopt double pump system, mobile phase A used: 30mmol/L ammonium acetate solution (takes 2.31g ammonium acetate in 1L water, mixing) B: acetonitrile, setting chromatogram determined wavelength is 280nm, and flow velocity is 1.0 mL min
-1; Sensitivity is 1.0 AUFS; Column temperature is 40 DEG C; Sample size is 10 μ L, respectively sample solution, contrast solution and blank solution is carried out sample determination;
Described assay step is with the residual quantity of external standard method calculation sample Eliquis.
2. high effective liquid chromatography for measuring Eliquis residual quantity according to claim 1, it is characterized in that: described mobile phase A is that 30mmol/L ammonium acetate solution (takes 2.31g ammonium acetate in 1L water, mixing), water is 2 redistilled waters, meets HPLC requirement.
3. high effective liquid chromatography for measuring Eliquis residual quantity according to claim 1, is characterized in that: Mobile phase B methanol solution is Chromatographic Pure Methanol.
4. high effective liquid chromatography for measuring Eliquis residual quantity according to claim 1, is characterized in that: described wavelength is 280nm, changes the maximum absorption wavelength that wavelength is Eliquis, and noiseless to solvent methanol etc.
5. high effective liquid chromatography for measuring Eliquis cleaning residual quantity according to claim 1, is characterized in that: detecting column temperature is 40 DEG C, effectively can improve peak type and effectively detect minimal residue.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105044269A (en) * | 2015-06-30 | 2015-11-11 | 成都百裕科技制药有限公司 | Method for detecting initial material II in apixaban through reversed-phase high performance liquid chromatography |
| CN107991412A (en) * | 2017-11-30 | 2018-05-04 | 江苏宝众宝达药业有限公司 | The method of high effective liquid chromatography for measuring Eliquis impurity content |
| CN109030655A (en) * | 2018-08-17 | 2018-12-18 | 无锡凯夫制药有限公司 | A kind of method of quick measurement Eliquis content |
| CN111521707A (en) * | 2020-05-11 | 2020-08-11 | 苏州必宜生物科技有限公司 | Method for determining apixaban concentration in blood plasma by LC-MS/MS |
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|---|---|---|---|---|
| CN102770126A (en) * | 2010-02-25 | 2012-11-07 | 百时美施贵宝公司 | Apixaban formulations |
| CN103833755A (en) * | 2014-03-24 | 2014-06-04 | 重庆东得医药科技有限公司 | Crystal form B of Apixaban and preparation method thereof |
| WO2014108919A2 (en) * | 2013-01-09 | 2014-07-17 | Msn Laboratories Limited | NOVEL INTERMEDIATE AND POLYMORPHS OF 1-(4-METHOXYPHENYL)-7-OXO-6-[4-(2-OXOPIPERIDIN-1-YL)PHENYL]-4,5,6,7-TETRAHYDRO-1H-PYRAZOLO[3,4-c] PYRIDINE-3-CARBOXAMIDE AND PROCESS THEREOF |
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2014
- 2014-10-30 CN CN201410596393.7A patent/CN104316637B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102770126A (en) * | 2010-02-25 | 2012-11-07 | 百时美施贵宝公司 | Apixaban formulations |
| WO2014108919A2 (en) * | 2013-01-09 | 2014-07-17 | Msn Laboratories Limited | NOVEL INTERMEDIATE AND POLYMORPHS OF 1-(4-METHOXYPHENYL)-7-OXO-6-[4-(2-OXOPIPERIDIN-1-YL)PHENYL]-4,5,6,7-TETRAHYDRO-1H-PYRAZOLO[3,4-c] PYRIDINE-3-CARBOXAMIDE AND PROCESS THEREOF |
| CN103833755A (en) * | 2014-03-24 | 2014-06-04 | 重庆东得医药科技有限公司 | Crystal form B of Apixaban and preparation method thereof |
Non-Patent Citations (4)
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105044269A (en) * | 2015-06-30 | 2015-11-11 | 成都百裕科技制药有限公司 | Method for detecting initial material II in apixaban through reversed-phase high performance liquid chromatography |
| CN107991412A (en) * | 2017-11-30 | 2018-05-04 | 江苏宝众宝达药业有限公司 | The method of high effective liquid chromatography for measuring Eliquis impurity content |
| CN109030655A (en) * | 2018-08-17 | 2018-12-18 | 无锡凯夫制药有限公司 | A kind of method of quick measurement Eliquis content |
| CN111521707A (en) * | 2020-05-11 | 2020-08-11 | 苏州必宜生物科技有限公司 | Method for determining apixaban concentration in blood plasma by LC-MS/MS |
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| CN104316637B (en) | 2016-08-17 |
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Address after: 226532 No. 10, Yuejiang Road, Changjiang town (Rugao port area), Rugao City, Nantong City, Jiangsu Province Patentee after: Jiangsu Baozhong Baoda Pharmaceutical Co.,Ltd. Address before: 226532 No. 10, Yuejiang Road, Changjiang town (Rugao port area), Rugao City, Nantong City, Jiangsu Province Patentee before: JIANGSU BAOZONG & BAODA PHARMACHEM Co.,Ltd. |