CN104316637B - High effective liquid chromatography for measuring Eliquis cleans residual quantity - Google Patents
High effective liquid chromatography for measuring Eliquis cleans residual quantity Download PDFInfo
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- CN104316637B CN104316637B CN201410596393.7A CN201410596393A CN104316637B CN 104316637 B CN104316637 B CN 104316637B CN 201410596393 A CN201410596393 A CN 201410596393A CN 104316637 B CN104316637 B CN 104316637B
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Abstract
The present invention relates to a kind of high effective liquid chromatography for measuring Eliquis and clean residual quantity, belong to chemical pharmacy detection field.It is an object of the invention to provide a kind of simple to operate, quick, measurement result Eliquis accurately and reliably cleans the assay method of residual, evaluates cleaning effect, to ensure drug quality and drug safety.This high effective liquid chromatography for measuring Eliquis of the present invention cleans residual quantity, simple to operate, quick, measurement result accurately and reliably, it is thus possible to valuator device cleaning performance effectively；The present invention have employed 280nm in the selection of flowing phase, effectively reduces detection limit；The present invention, on chromatogram column temperature selects, uses 40 DEG C, it is possible to be effectively improved peak type and effectively detect minimal residue, selects methyl alcohol in the selection of solvent, and this wavelength of methyl alcohol is without absorbing, it is to avoid the solvent interference to detection, improves the reliability of detection method.
Description
Technical field
The present invention relates to a kind of high effective liquid chromatography for measuring Eliquis and clean residual quantity, belong to chemistry
Pharmacy detection field.
Background technology
The problem inevitably relating to total equipment in production of raw medicine technique, switches product in total link, as
Really equipment cleans not exclusively, and the last material residual produced will necessarily be brought in the material next time produced, due to medicine poison
Property difference, the size of residual quantity can directly influence Drug safety and drug quality, therefore control to clean residual and reach to close
Reason limit is unusual necessity, and is also the GMP laws and regulations requirement as minimum pharmaceutical requirements.General, product allows
Impurity exists in the range of safety allows, and therefore, uses Eliquis in certain instrument analytical method detection product
Clean residual evaluation cleaning performance and drug safety is the most necessary, be also the most urgent.
At present, the domestic detection method not yet having pharmacopeia and pertinent literature to propose this material, to the detection of minimal residue more
Uncorrelated report.
For Eliquis residual quantity detection, relying on chemistry or general ultraviolet equipment is to be extremely difficult to trace ppm level
Other testing requirement, the most also to ensure the good range of linearity, good stability, preferable accuracy and precision, and this grinds
The method studied carefully can be simple to operate, quick, measurement result accurately and reliably, such that it is able to effectively control the residual of Eliquis
Amount, it is ensured that medicinal safety and reliability.
Therefore, this research uses HPLC method, establishes a kind of Eliquis and cleans the assay method of residual quantity.
Summary of the invention
It is an object of the invention to provide a kind of simple to operate, quick, measurement result Eliquis accurately and reliably cleans residual
Assay method, 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: bag
Include 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: weigh Eliquis reference substance 25mg in 50mL measuring bottle, uses
Diluent dissolves, and is settled to scale, filters to obtain contrast solution；
(3) contrast solution (0.0005mg/mL) preparation: precision measures comparison stock solution 1.0mL in 100mL measuring bottle, fixed
Hold to scale, shake up；Precision measures above-mentioned solution 1mL in 10mL measuring bottle again, is settled to scale, shakes up；
Described sample determination step is the look selecting model to be Shimadzu Inertsil ODS-3V 5 μm 250 × 4.6mm
Spectrum post, uses double pump system, mobile phase A used: 30mmol/L ammonium acetate solution (weigh 2.31g ammonium acetate in 1L water,
Mixing) B: acetonitrile, setting chromatogram detection wavelength is 1.0 mLmin as 280nm, flow velocity-1；Sensitivity is 1.0 AUFS；Column temperature is
40℃；Sample size is 10 L, respectively sample solution, contrast solution and blank solution is carried out sample determination；
Described assay step is the residual quantity calculating sample Eliquis with external standard method.
Further, described mobile phase A be 30mmol/L ammonium acetate solution (weigh 2.31g ammonium acetate in 1L water,
Mixing), 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 to solvent methanol
Etc. noiseless.
Further, detection column temperature is 40 DEG C, it is possible to is effectively improved peak type and effectively detects minimal residue.
Beneficial effect: this high effective liquid chromatography for measuring Eliquis of the present invention cleans residual quantity, simple to operate, fast
Speed, measurement result accurately and reliably, it is thus possible to valuator device cleaning performance effectively；The present invention have employed in the selection of flowing phase
280nm, effectively reduces detection limit；The present invention, on chromatogram column temperature selects, uses 40 DEG C, it is possible to is effectively improved peak type and has
Effect detection minimal residue, selects methyl alcohol in the selection of solvent, and this wavelength of methyl alcohol is without absorbing, it is to avoid the solvent interference to detection,
Improve the reliability of detection method.
Accompanying drawing explanation
It is further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 is that specificity of the present invention tests collection of illustrative plates.
Fig. 2 is precision test collection of illustrative plates.
Fig. 3 is detection limit collection of illustrative plates.
Fig. 4 be contrast solution be the collection of illustrative plates of 0.00005.
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%.
Detailed description of the invention
Following enforcement row can make those skilled in the art be more completely understood by the present invention, but the most therefore by the present invention
It is limited among described scope of embodiments.
Embodiment 1
High effective liquid chromatography for measuring Eliquis cleaning residual method:
Instrument and reagent: LC 2010A high performance liquid chromatograph (quaternary pump, degassing unit, UV detector, post case, automatically
Injector, system monitor, LCsolution) (Shimadzu Corporation of Japan)；Test sample (manufacturer: Jiangsu treasured crowd's treasured reaches medicine company
Co., Ltd, acetonitrile, methyl alcohol (merck chromatographically pure), other reagent analysis is pure, and water is for making fresh redistilled water by oneself.
Chromatography condition: chromatographic column: Inertsil ODS-3V 5 μm 250 × 4.6mm；Mobile phase A: 30mmol/L
Ammonium acetate solution, Mobile phase B: acetonitrile, isocratic elution；Detection wavelength: 280nm；Computational methods: external standard method；Flow velocity: 1.0
mLmin-1；Sensitivity: 1.0 AUFS；Sample size: 10 L；Column temperature 40 DEG C.
Specificity is tested: sample introduction equipment drip washing methyl alcohol, contrast solution sample methyl alcohol and contrast solution, investigation side respectively
The specificity of method, confirms that the blank mensuration to Eliquis of this verification method does not exist interference；Every day, sample introduction contrast solution, confirmed
Retention time and the system suitability of Eliquis meet acceptable standard.In blank solution noiseless；Main peak in contrast solution
Signal to noise ratio be not less than 30.
Solution is prepared:
Contrast solution stock solution (0.5mg/mL): take Eliquis reference substance 25mg in 50mL measuring bottle, molten with diluent
Solve, be settled to scale, shake up.
Contrast solution (0.0005mg/mL): precision measure comparison stock solution 1.0mL in 100mL measuring bottle, be settled to carve
Degree, 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: take direct injected after whole washing lotion filters
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: take contrast solution, continuous sample introduction 6 times, calculate the retention time of Eliquis and peak area
RSD.The RSD of actually measured retention time is 0.03%；The RSD of peak area is 0.59%
Method precision: repeat to prepare 6 parts of contrast solutions, every part of solution sample introduction 1 time, the peak area of Eliquis/title sample
The RSD of amount is 0.68%.
Intermediate precision: analyzed personnel by another name, in another sky, repetition methods precision test on another instrument,
The RSD of the peak area/sample weighting amount of Eliquis is 6.87%.
Showing that reappearance is good, method is reliable.
Embodiment 3
The detection limit test of the inventive method:
By the Eliquis reference substance solution of sample introduction low concentration, when signal to noise ratio (S/N) is 3, this concentration is its detection
Limit, detection limit solution sample introduction 3 pin, calculate and report the RSD of peak area.Finally determine concentration when S/N is about 2.8
0.0125ug/ml, for detection limit.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 is 10, this concentration is its quantitative limit, quantitatively
Limit solution sample introduction 6 pin, calculates and reports the RSD of peak area.When finally determining S/N about 9.4,0.05ug/ml is quantitative limit,
Area RSD is 3.3%.
Embodiment 5
The accuracy test of the inventive method.
By the cleaning condition that simulation is actual, detect the concentration of Eliquis in leacheate by the method for inspection of empirical tests.
Optional quantitative limit, 50%, 100%, 150% 4 concentration, each concentration repeats 3 times, the rate of recovery of checking sampling method
And repeatability, to verify that the sampling method of leacheate can meet the requirements.
Eliquis stock solution: weigh Eliquis reference substance 25mg in 50mL measuring bottle, dissolves with diluent, fixed
Hold, shake up.
0.005mg/ml Eliquis solution: precision measure comparison stock solution 1.0mL in 100mL measuring bottle, be settled to carve
Degree, shakes up.
50% concentration:
Preparing the glass round bottom flask of a clean 50mL, precision measures the Eliquis of the 0.005mg/mL of 1.5mL
Methanol solution, as far as possible uniform drenches the inwall at round-bottomed flask, allows round-bottomed flask natural drying.30mL is added in round-bottomed flask
Methyl alcohol, and be warming up to backflow, be incubated at least 1 hour.It is cooled to stop backflow, directly takes leacheate.Repeat above operation 3
Secondary, take 3 parts of leacheates.
100% concentration:
Preparing the glass round bottom flask of a clean 50mL, precision measures the Eliquis of the 0.005mg/mL of 3.0mL
Methanol solution, as far as possible uniform drenches the inwall at round-bottomed flask, allows round-bottomed flask natural drying.30mL is added in round-bottomed flask
Methyl alcohol, and be warming up to backflow, be incubated at least 1 hour.It is cooled to stop backflow, directly takes leacheate.Repeat above operation 3
Secondary, take 3 parts of leacheates.
150% concentration:
Preparing the glass round bottom flask of a clean 50mL, precision measures the Eliquis of the 0.005mg/mL of 4.5mL
Methanol solution, as far as possible uniform drenches the inwall at round-bottomed flask, allows round-bottomed flask natural drying.30mL is added in round-bottomed flask
Methyl alcohol, and be warming up to backflow, be incubated at least 1 hour.It is cooled to stop backflow, directly takes leacheate.Repeat above operation 3
Secondary, take 3 parts of leacheates.
Quantitative limit: measure under item according to quantitative limit, determine the concentration of solution.
Detecting the concentration of Eliquis in leacheate by the method for inspection of empirical tests, after system stability, sample introduction blank is molten
Liquid, Eliquis standard liquid, leacheate solution, calculate the concentration of 12 samples, and calculate the rate of recovery respectively.
The actual recovered rate of quantitative limit level is 88.9, and the rate of recovery of 50% level is 90.8%, the rate of recovery of 100% level
Being 92.3%, the rate of recovery of 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.Below
Time point need to be investigated: 0,3h, 6h, 24h and 48h, if it find that solution is unstable, need to shorten time interval.Solution appearance should
Clarification；It is≤0.89% with the maximum deviation of contrast solution main peak area during 0h, is i.e. considered as solution-stabilized.
Claims (1)
1. the method for Eliquis residual quantity in high effective liquid chromatography for measuring cleaning fluid, it is characterised in that: include sample preparation,
Sample determination and cubage step；
Described sample preparation step is as follows:
(1) blank solution preparation: use solvent for cleaning methyl alcohol preparation blank solution；
(2) contrast solution stock solution preparation: weigh Eliquis reference substance 25mg in 50mL measuring bottle, dissolves with diluent, fixed
Holding to scale, filter to obtain contrast solution stock solution, concentration is 0.5mg/mL；
(3) contrast solution preparation: precision measures contrast solution 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, obtains contrast solution, and concentration is 0.0005mg/
mL；
Described sample determination step is the chromatographic column selecting model to be Shimadzu Inertsil ODS-3V 5 μm 250 × 4.6mm,
Use double pump system, mobile phase A used: 30mmol/L ammonium acetate solution, Mobile phase B: acetonitrile, the compound method of mobile phase A
For weighing 2.31g ammonium acetate in 1L water, mixing；Setting chromatogram detection wavelength is 1.0mL min as 280nm, flow velocity-1；Sensitive
Degree is 1.0AUFS；Column temperature is 40 DEG C；Sample size is 10 μ L；
Described assay step for carry out sample determination respectively by sample solution, contrast solution and blank solution, with external standard method
Calculate the residual quantity of sample Eliquis.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105044269B (en) * | 2015-06-30 | 2016-08-24 | 成都百裕制药股份有限公司 | The method of starting material II in reversed-phase high-performance liquid chromatography detection Eliquis |
| 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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| 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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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. |
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