CN103743832B - HPLC (High Performance Liquid Chromatography) measuring method of related substances in Dabigatran - Google Patents
HPLC (High Performance Liquid Chromatography) measuring method of related substances in Dabigatran Download PDFInfo
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Abstract
The invention discloses a detection method of related substances in Dabigatran. The detection method adopts a diode array detector and takes an acetonitrile (flowing phase A)-phosphate solution (flowing phase B) as a flowing phase. The detection method comprises the steps of adding the flowing phase to prepare a solution containing 0.5mg of Dabigatran in 1ml by taking the Dabigatran and an appropriate quantity of related preparation containing the Dabigatran to be used as a test solution, diluting the test solution into solution containing 5mu g of Dabigatran in 1ml by using the flowing phase to be used as a contrast solution, respectively injecting a sample, wherein the sum of all impurity peak areas in a chromatogram map of the test solution is not greater than a main peak area of the contrast solution. The detection method of the Dabigatran and the related preparation containing the Dabigatran provided by the invention have the advantages that the impurities of the Dabigatran and the degradation product condition are rapidly and accurately detected, the operation is simple and direct, the sensitivity is high, and the product quality can be well controlled.
Description
Technical field
The present invention relates to the related substance detection method of a kind of dabigatran and the related preparations containing dabigatran, belong to Pharmaceutical Analysis detection field.
Background technology
Dabigatran etcxilate is direct thrombin inhibitor, have can oral, potent, without the need to features such as special Medication monitor, drug interaction are few.External, in vivo studies and clinical every research all point out dabigatran etcxilate to have good curative effect and pharmaco-kinetic properties, and potential applicability in clinical practice is optimistic, and it successfully goes on the market is an important breakthrough of anticoagulation medicine research field.Because dabigatran etcxilate likely introduces retained material and other related substance in building-up process, in storage, also likely produce catabolite, this law effectively carries out purity analysis to the dabigatran after dissociating, and this test adopts common chromatographic column (C
18chromatographic column) achieve the mensuration of dabigatran related substance fast and accurately, ensure that the quality controllable of dabigatran, have important practical significance in the quality control of synthesis and production process.Not yet retrieve the assay method of the HPLC of dabigatran related substance at present.The related substance detection method of dabigatran of the present invention and the related preparations containing dabigatran, can detect impurity and the catabolite situation of dabigatran fast and accurately.Simple to operation, highly sensitive, product quality can be controlled preferably.
Summary of the invention
The object of the present invention is to provide a kind of diode array detector that adopts to dabigatran and the assay method carrying out Related substances separation containing the related preparations of dabigatran.
The realization of Related substances separation method of the present invention comprises the following steps:
(1) chromatographic condition: detecting device adopts diode array detector, column temperature 30 DEG C, flow velocity 1.0ml/min.The mobile phase of chromatographic condition is made up of acetonitrile (mobile phase A)-phosphate solution (Mobile phase B), wherein phosphate solution (Mobile phase B), concentration range 0.005 ~ 0.015mol/L, adjustment pH scope 6.5 ~ 8.5, its optium concentration is 0.01mol/L, and optimal pH is about 7.6.Chromatogram column temperature is 30 DEG C, flow velocity 1.0ml/min.Theoretical cam curve calculates by dabigatran peak and is not less than 3000.
(2) preparation of need testing solution:
Get dabigatran or the related preparations containing dabigatran is appropriate, add the solution that mobile phase is mixed with 0.5mg/ml, as need testing solution.
(3) preparation of contrast solution:
Measure above-mentioned (2) need testing solution appropriate, add mobile phase and be diluted to the solution about containing 5 μ g in every 1ml, solution in contrast.
(4) assay method:
Get contrast solution 10 μ l injection liquid chromatography, regulate detection sensitivity, major component chromatogram peak height is made to be about 20% of full scale, get need testing solution 10 μ l injection liquid chromatography again, record chromatogram is to 2 times of dabigatran main peak retention time, if any impurity peaks in need testing solution chromatogram, measure each impurity peak area and, the peak area (see accompanying drawing 1, accompanying drawing 2) of main peak in contrast solution must not be greater than.
The related substance detection method of dabigatran of the present invention and the related preparations containing dabigatran, the preparation of need testing solution in above-mentioned (2): for meeting the needs of need testing solution detectable concentration, get dabigatran appropriate, being mixed with concentration with mobile phase is the solution of every 1ml containing 0.5mg, as need testing solution.Be diluted to the solution of a series of variable concentrations more respectively with mobile phase, respectively sample introduction 10 μ l, make it to produce the signal that main peak is baseline noise three times.Through test, detectability is 0.5ng(S/N >=3), if calculate with concentration 0.5mg/ml during Related substances separation, its limit of detection is 0.1%.
The related substance detection method of dabigatran of the present invention and the related preparations containing dabigatran, the preparation of contrast solution in above-mentioned (3): precision measures above-mentioned need testing solution 1ml, put in 100ml measuring bottle, add mobile phase and dissolve and be diluted to scale, solution in contrast.When this contrast solution concentration range is in 1 ~ 8 μ g/ml, peak area and concentration are good linear relationship, and its linearly dependent coefficient is that 0.9997(is shown in accompanying drawing 3).
Dabigatran of the present invention and the related substance detection method of related preparations containing dabigatran, its related substance detects in high-efficient liquid phase chromatogram, occurs if any impurity peaks, measure each impurity peak area and, the main peak area (1%) of contrast solution should be greater than.
Dabigatran of the present invention and the related substance detection method of related preparations containing dabigatran, be applicable to variously need to carry out the dabigatran of related substance detection and the related preparations containing dabigatran, as dabigatran etcxilate sheet.
The present invention's good effect is compared with the prior art:
1, the present invention uses diode array detector to carry out related substance detection to this product, establish a kind of effective ways of evaluation chromatographic peak purity of quicklook: the purity that can judge chromatographic peak intuitively, and judge the position that there is Interference Peaks, whole spectral informations of " at stream " can be obtained, obtain the absorption spectrum of chromatographic component fast, the UV-visible detector that diode array detector is more general, it is qualitative that advantage is to rely on the retention time of chromatogram to carry out, and the spectral information that can provide according to it carries out qualitative, substantially increase confidence level qualitatively.And effectively improve its detection sensitivity.
2, the related substance detection method of dabigatran of the present invention and the related preparations containing dabigatran, can detect impurity and the catabolite situation of dabigatran fast and accurately.Product quality can be controlled preferably.
3, the present invention is applicable to the dabigatran of various formulation and the related preparations containing dabigatran, as dabigatran sheet.
4, detection method is accurate, simple to operation, and favorable reproducibility is highly sensitive, fully can meet the requirement that Related substances separation measures with decomposition product, the special impurities preferably in Quality control, guarantee product quality, practical at work.
Embodiment
embodiment one:
1, chromatographic condition and system suitability:
1.1, the selection of chromatographic condition:
Instrument: Shimadzu: LC-20AT, SPD-M20A, SIL-20A, DGU-20A5, its optimum column temperature 30 DEG C, flow velocity 1.0ml/min.Liquid-phase chromatographic column octadecylsilane chemically bonded silica is filling agent (250mm × 4.6mm, 5 μm), with reference to pertinent literature binding tests concrete condition, successively select the multiple condition such as methanol-water, acetonitrile-water, methanol-acetonitrile-water, methanol-acetonitrile-phosphate solution, finally determine that mobile phase is made up of acetonitrile (mobile phase A)-phosphate solution (Mobile phase B), wherein phosphate solution (Mobile phase B), concentration range 0.005 ~ 0.015mmol/L, adjustment pH scope 6.5 ~ 8.5, its optium concentration is 0.01 mol/L, and optimal pH is about 7.6.Sample size 10 μ l.
Under this chromatographic condition, dabigatran main peak retention time is moderate, and peak shape is better.
1.2, to get dabigatran reference substance appropriate for sensitivity determination, being mixed with concentration with mobile phase is the solution of every 1ml containing 0.5mg, be diluted to the solution of a series of variable concentrations more respectively with mobile phase, respectively sample introduction 10 μ l, make it to produce the signal that main peak is baseline noise three times.Through test, detectability is 0.5ng(S/N >=3), if calculate with concentration 0.5mg/ml during Related substances separation, its limit of detection is 0.1%.Result proves, the method is highly sensitive, fully can meet the requirement that Related substances separation measures.
1.3, stability of solution is got with a need testing solution, respectively at 0,2,6,10,15 hour respectively sample introduction measure, its main peak peak area and determination of related substances result basicly stable in 15 hours.
Above test findings shows, the method is easy to be sensitive, favorable reproducibility, can detect preferably the quality of dabigatran in sample preferably.
2, the preparation of need testing solution:
Get dabigatran or the related preparations containing dabigatran is appropriate, add the solution that mobile phase is mixed with 0.5mg/ml, as need testing solution.
3, the preparation of contrast solution:
Measure above-mentioned (2) need testing solution appropriate, add mobile phase and be diluted to the solution about containing 5 μ g in every 1ml, solution in contrast.
4, the detection of dabigatran and the related preparations related substance containing dabigatran:
Get contrast solution 10 μ l injection liquid chromatography, regulate detection sensitivity, major component chromatogram peak height is made to be about 20% of full scale, get need testing solution 10 μ l injection liquid chromatography again, record chromatogram is to 2 times of dabigatran main peak retention time, if any impurity peaks in need testing solution chromatogram, measure each impurity peak area and, the peak area of main peak in contrast solution must not be greater than.
the mensuration of embodiment two, dabigatran etcxilate sheet related substance
Get this product 20, accurately weighed, porphyrize, precision takes in right amount (being about equivalent to dabigatran 5mg), adds mobile phase and is mixed with the solution containing 0.5mg in every 1ml, filter, get filtrate as need testing solution.Precision measures in right amount, adds mobile phase and is diluted to the solution about containing 5 μ g in every 1ml, solution in contrast.Under following selected chromatographic condition: diode array detector (Shimadzu: LC-20AT, SPD-M20A, SIL-20A, DGU-20A5), be filling agent with octadecylsilane chemically bonded silica, mobile phase is made up of acetonitrile (mobile phase A)-phosphate solution (Mobile phase B), wherein phosphate solution (Mobile phase B), concentration range 0.005 ~ 0.015mol/L, adjustment pH scope 6.5 ~ 8.5, its optium concentration is 0.01mol/L, and optimal pH is about 7.6.Chromatogram column temperature is 30 DEG C, flow velocity 1.0ml/min.
Theoretical cam curve should be not less than 3000. by the calculating of dabigatran peak and get contrast solution 10 μ l injection liquid chromatography, regulate detection sensitivity, make that contrast solution main peak is high is about 20% of full scale, get each 10 μ l of need testing solution again, injection liquid chromatography respectively, record chromatogram is to 2 times of major component peak retention time.If any impurity peaks in need testing solution chromatogram, measure each impurity peak area and, the main peak area of contrast solution must not be greater than.
Table 1. dabigatran etcxilate sheet determination of related substances result
| First second batch of lot number the 3rd batch |
| Related substance (%) 0.18 0.17 0.21 |
the mensuration of embodiment three, dabigatran etcxilate capsule related substance
Get this product content appropriate, porphyrize, precision takes fine powder (being about equivalent to dabigatran 5mg), adds mobile phase and is mixed with the solution containing 0.5mg in every 1ml, filter, get filtrate as need testing solution.Precision measures in right amount, adds mobile phase and is diluted to the solution about containing 5 μ g in every 1ml, solution in contrast.Under following selected chromatographic condition: diode array detector (Shimadzu: LC-20AT, SPD-M20A, SIL-20A, DGU-20A5), be filling agent with octadecylsilane chemically bonded silica, mobile phase is made up of acetonitrile (mobile phase A)-phosphate solution (Mobile phase B), wherein phosphate solution (Mobile phase B), concentration range 0.005 ~ 0.015mol/L, adjustment pH scope 6.5 ~ 8.5, its optium concentration is 0.01mol/L, and optimal pH is about 7.6.Chromatogram column temperature is 30 DEG C, flow velocity 1.0ml/min.
Theoretical cam curve should be not less than 3000. by the calculating of dabigatran peak and get contrast solution 10 μ l injection liquid chromatography, regulate detection sensitivity, make that contrast solution main peak is high is about 20% of full scale, get each 10 μ l of need testing solution again, injection liquid chromatography respectively, record chromatogram is to 2 times of major component peak retention time.If any impurity peaks in need testing solution chromatogram, measure each impurity peak area and, the main peak area of contrast solution must not be greater than.
Table 2. dabigatran etcxilate capsule determination of related substances result
| First second batch of lot number the 3rd batch |
| Related substance (%) 0.22 0.15 0.13 |
the mensuration of embodiment four, dabigatran etcxilate sustained release tablets related substance
Get this product appropriate, porphyrize, precision takes fine powder (being about equivalent to dabigatran 5mg), adds mobile phase and is mixed with the solution containing 0.5mg in every 1ml, filter, get filtrate as need testing solution.Precision measures in right amount, adds mobile phase and is diluted to the solution about containing 5 μ g in every 1ml, solution in contrast.Under following selected chromatographic condition: diode array detector (Shimadzu: LC-20AT, SPD-M20A, SIL-20A, DGU-20A5), be filling agent with octadecylsilane chemically bonded silica, mobile phase is made up of acetonitrile (mobile phase A)-phosphate solution (Mobile phase B), wherein phosphate solution (Mobile phase B), concentration range 0.005 ~ 0.015mol/L, adjustment pH scope 6.5 ~ 8.5, its optium concentration is 0.01mol/L, and optimal pH is about 7.6.Chromatogram column temperature is 30 DEG C, flow velocity 1.0ml/min.
Theoretical cam curve should be not less than 3000. by the calculating of dabigatran peak and get contrast solution 10 μ l injection liquid chromatography, regulate detection sensitivity, make that contrast solution main peak is high is about 20% of full scale, get each 10 μ l of need testing solution again, injection liquid chromatography respectively, record chromatogram is to 2 times of major component peak retention time.If any impurity peaks in need testing solution chromatogram, measure each impurity peak area and, the main peak area of contrast solution must not be greater than.
Table 3. dabigatran etcxilate sustained release tablets determination of related substances result
| First second batch of lot number the 3rd batch |
| Related substance (%) 0.18 0.15 0.23 |
accompanying drawing illustrates:
Fig. 1: the HPLC collection of illustrative plates of contrast solution
Fig. 2: the HPLC collection of illustrative plates of need testing solution
Fig. 3: dabigatran l.o.i linear relationship chart.
Claims (4)
1. a related substance detection method for dabigatran and the related preparations containing dabigatran, is characterized in that detecting device adopts diode array detector; The mobile phase of chromatographic condition is made up of mobile phase A acetonitrile and Mobile phase B phosphate solution, wherein the concentration range of phosphate solution is 0.005 ~ 0.015mol/L, adjustment pH scope 6.5 ~ 8.5, and chromatogram column temperature is 30 DEG C, flow velocity 1.0ml/min, gradient elution program is as following table:
, detection method comprises the following steps:
(1) chromatographic condition:
This product adopts octadecylsilane chemically bonded silica to be filling agent, and with acetonitrile-phosphate solution for mobile phase, flow velocity is 1.0ml/min, and theoretical cam curve calculates by dabigatran and is not less than 3000;
(2) preparation of need testing solution:
Get dabigatran or the related preparations containing dabigatran is appropriate, add the solution that mobile phase is mixed with 0.5mg/ml, as need testing solution;
(3) preparation of contrast solution:
Measure above-mentioned (2) need testing solution appropriate, add mobile phase and be diluted to the solution about containing 5 μ g in every 1ml, solution in contrast;
(4) assay method:
Get contrast solution 10 μ l injection liquid chromatography, regulate detection sensitivity, major component chromatogram peak height is made to be about 20% of full scale, get need testing solution 10 μ l injection liquid chromatography again, record chromatogram is to 2 times of dabigatran main peak retention time, if any impurity peaks in need testing solution chromatogram, measure each impurity peak area and, the peak area of main peak in contrast solution must not be greater than.
2. the related substance detection method of dabigatran according to claim 1 and the related preparations containing dabigatran, is characterized in that: the optium concentration of Mobile phase B phosphate solution is 0.01mol/L, and optimal pH is 7.6.
3. the detection method of dabigatran according to claim 1 and the related substance containing dabigatran related preparations, it is characterized in that: the wherein preparation of (3) contrast solution: get dabigatran related preparations appropriate, add the solution that mobile phase is mixed with 0.5mg/ml, as need testing solution; Precision measures test sample 1ml again, puts in 100ml measuring bottle, adds mobile phase and dissolves and be diluted to scale, solution in contrast.
4. according to the detection method of described dabigatran arbitrary in claims 1 to 3 and the related substance containing dabigatran related preparations, it is characterized in that: the method be applicable to various need to carry out related substance detection dabigatran or related preparations containing dabigatran, as tablet, capsule, granule, parenteral solution, controlled release ease up the compound preparation of controlled release and various dabigatran.
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