CN106770849B - A kind of detection method measuring doxercalciferol and its impurities - Google Patents
A kind of detection method measuring doxercalciferol and its impurities Download PDFInfo
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Abstract
The present invention provides the detection method of a kind of measurement doxercalciferol and its impurities, detection method is using highly effective liquid phase chromatography detection method, the chromatographic condition of detection method：Chromatographic column：Using globular preteins hydrophilic silica gels or octadecylsilane chemically bonded silica as stationary phase；Mobile phase：The mixed solution of acid solution and organic solvent composition；Flow velocity：1.5~2.0mL/min；Detection wavelength：210nm；Column temperature：Room temperature；Sample size：5~10uL；Sample concentration：3~5mg/mL；The volume ratio of acid solution and organic solvent in mixed solution lowers with the elution progress gradient of high performance liquid chromatography.In the present invention, during the gradient elution of highly effective liquid phase chromatography detection method, the volume ratio of acid solution and organic solvent in mobile phase lowers with the elution progress gradient of high performance liquid chromatography, it realizes while to the separation and detection of doxercalciferol and its contained three kinds of impurity, the efficiency of separation and detection is improved, and reduces detection time and the relative retention time of main peak.
Description
Technical field
The present invention relates to chemicals detection technique field more particularly to a kind of inspections measuring doxercalciferol and its impurities
Survey method.
Background technology
Doxercalciferol (Doxercalciferol) is also known as 1-A hydroxy vitamin D2s, is that a kind of calciferol analog is main
Parathyroid gland is acted on, the secretion of intact PTH, lesser degree can effectively be inhibited to increase blood calcium and serium inorganic phosphorus concentration,
The new drugs such as the SHPT for FDA approvals for osteoporosis, 3 to 4 grades of Patients with Chronic Renal Disease.The molecular formula of doxercalciferol is：
C28H44O2, chemical structural formula is as follows：
In doxercalciferol building-up process, the by-product generated in some relevant impurity substances such as production process will produce
Or intermediate or catabolite etc..These impurity may be due to removing purity and quality incomplete and that influence drug.Currently,
Known impurity has 3 kinds, respectively：Related substance 1, related substance 2, related substance 3.The chemical structural formula of above-mentioned three kinds of impurity
As follows：
For introduced in doxercalciferol synthesis technology above-mentioned impurity (or be referred to as " by-product, intermediate and correlation
Degradation impurity ") be required for carrying out quality control in doxercalciferol raw material and preparation.Therefore, it realizes to miscellaneous in doxercalciferol
The separation of matter and measurement have great importance to the production and storage of doxercalciferol raw material and preparation.
Have a Li Jing for analyzing detecting method of the doxercalciferol in relation to substance at present, season gold just exist《Chinese Journal
of Analysis Laboratory》Mistake reported in the o. 11th of volume 28.It uses octadecylsilane chemically bonded silica for filling
Agent carries out gradient elution, flow velocity 1.7ml/min, Detection wavelength 205nm using water and acetonitrile as mobile phase, and column temperature is 35 DEG C,
Sampling volume is 5 μ L.The average recovery rate and Intermediate precision of this method are higher.But the document is not described the party
Method is to the separation detection abilities of above-mentioned related impurities, and the relative retention time of main peak under this methodology is longer.
In addition, Ninus Simonzadeh et al. exist《Determination of Degradation Products of
Doxercalciferol by Solid-Phase Extraction and Reversed-Phase HPLC》Reported in one text
It crosses, gradient elution, flow velocity 1.6ml/min, Detection wavelength 274nm is carried out as mobile phase using water and acetonitrile, column temperature is 40 DEG C,
Sampling volume is 100 μ L.The document realizes separation detection to above-mentioned related substance 1, related substance 2, related substance 3.But
The detection time of this method is longer, and the relative retention time of main peak is longer, and the impurity response at 274nm wavelength is relatively low,
Keep the impurity content measured more relatively low than practical, is unfavorable for the control of impurity in doxercalciferol raw material, to degree of eventually affecting bone
Change the therapeutic effect of alcohol formulations clinically.
Invention content
It is an object of the invention to disclose a kind of detection method measuring doxercalciferol and its impurities, to realize to degree
The separation and detection of four kinds of substances can be achieved at the same time in ostelin and its contained three kinds of separation and detection in relation to substance, improves and divides
From the efficiency with detection, and reduce detection time and the relative retention time of main peak.
To achieve the above object, the present invention provides the detection method of a kind of measurement doxercalciferol and its impurities, institutes
Detection method is stated using highly effective liquid phase chromatography detection method, the chromatographic condition of the detection method is：
Chromatographic column：Using globular preteins hydrophilic silica gels or octadecylsilane chemically bonded silica as stationary phase；
Mobile phase：The mixed solution of acid solution and organic solvent composition；
Flow velocity：1.5~2.0mL/min；
Detection wavelength：210nm；
Column temperature：40℃；
Sample size：5~10uL；
Sample concentration：3~5mg/mL；
The volume ratio of acid solution and organic solvent in the mixed solution with high performance liquid chromatography elution progress
Gradient lowers.
As a further improvement on the present invention, further include using organic solvent sample dissolution.
As a further improvement on the present invention, the acid in the acid solution is selected from phosphoric acid, formic acid, acetic acid, citric acid or three
One or two kinds of mixture with arbitrary proportion in fluoroacetic acid.
As a further improvement on the present invention, a concentration of 0.01~0.05vol% of the acid solution.
As a further improvement on the present invention, a concentration of 0.05vol% of the acid solution.
As a further improvement on the present invention, the organic solvent is selected from methanol or acetonitrile.
As a further improvement on the present invention, the volume ratio of the acid solution in the mixed solution and organic solvent is：
The volume ratio of 0min, acid solution and organic solvent is 30:70；
The volume ratio of 15min, acid solution and organic solvent is 28:72；
The volume ratio of 20min, acid solution and organic solvent is 15:85；
The volume ratio of 25min, acid solution and organic solvent is 15:85；
The volume ratio of 30min, acid solution and organic solvent is 0:100.
Compared with prior art, the beneficial effects of the invention are as follows：In the present invention, in the ladder of highly effective liquid phase chromatography detection method
It spends in elution process, the volume ratio of acid solution and organic solvent in mobile phase is terraced with the elution progress of high performance liquid chromatography
Degree lowers, and realizes while to the separation and detection of doxercalciferol and its contained three kinds of impurity, improves separation and detection
Efficiency, and reduce detection time and the relative retention time of main peak.
Description of the drawings
Fig. 1 is that the present invention measures doxercalciferol and its high performance liquid chromatography of impurities is carrying out height to sample solution
Effect liquid phase chromatogram detection is formed by chromatogram.
Specific implementation mode
The present invention is described in detail for each embodiment shown in below in conjunction with the accompanying drawings, but it should explanation, these
Embodiment is not limitation of the present invention, those of ordinary skill in the art according to function, method made by these embodiments,
Or the equivalent transformation in structure or replacement, all belong to the scope of protection of the present invention within.
Unless there is specified otherwise in specification, it is pure that the component in each embodiment in the present invention, raw material are all made of analysis
Rank.In addition, " g " in each embodiment is unit of weight " gram "；" h " is chronomere's " hour "；" ml " is volume unit " milli
It rises "；" room temperature " is 23 DEG C." vol% " is concentration unit " percent by volume ".
A kind of high efficiency liquid chromatography for separating and determining doxercalciferol and the method in relation to substance, the high performance liquid chromatography point
It is octadecylsilane chemically bonded silica for filler to analyse the chromatographic column that uses in the process, with acid solution and organic solvent in certain ratio
Mobile phase is mixed into be eluted.The one kind of acid in phosphoric acid, formic acid, acetic acid, citric acid or trifluoroacetic acid in acid solution
Or two kinds of mixtures with arbitrary proportion, and most preferably trifluoroacetic acid.Organic solvent is one kind in methanol or acetonitrile
Or the mixture of two kinds of arbitrary proportions, and most preferably acetonitrile.
Method of the present invention is suitable for separation and the measurement of three kinds of above-mentioned impurity, can be not only used for individually detecting a certain
Kind impurity, also can detach and detect simultaneously these three impurity, compared with prior art, method of the invention is faster, improves
The detection efficiency of highly effective liquid phase chromatography detection method.
Method of the present invention, in a specific embodiment.It is realized according to following technical scheme.Degree of taking is ossified
Alcohol, related substance 1, related substance 2,3 reference substance of related substance (being the pure rank of analysis) are each appropriate, using acetonitrile as organic
Solvent dissolves, and is containing 100 μ g of doxercalciferol, related substance 1, in relation to 3 each 1 μ g of substance 2 and related substance to be configured to every lml
System applicability solution.
Doxercalciferol reference substance is taken, is dissolved with acetonitrile, the reference substance solution that every lml contains 0.1 μ g is configured to.
It takes sample to be tested appropriate, is dissolved with acetonitrile, be configured to the test solution of every lml 100 μ g containing doxercalciferol.
System suitability solution, test solution and reference substance solution are taken respectively, are injected liquid chromatograph, are carried out efficient liquid
Phase chromatography detects.Chromatographic condition in the present embodiment is as follows.
The model of high performance liquid chromatograph：Dinoex U3000 8053386.Column diameter is 4.6mm.Chromatographic column：
Octadecylsilane chemically bonded silica is stationary phase；Mobile phase：The mixed solution of acid solution and organic solvent composition；Flow velocity：1.5~
2.0mL/min；Detection wavelength：210nm；Column temperature：40℃；Sample size：5~10uL；Sample concentration：3~5mg/mL；Column length：
150mm；The grain size of octadecylsilane chemically bonded silica particle is 5 μm.
Gradient elution is carried out using the mixed solution that the trifluoroacetic acid of 0.05vol% and acetonitrile are formed as mobile phase.It is mixed
The volume ratio for closing the acid solution and organic solvent in solution lowers with the elution progress gradient of high performance liquid chromatography, specifically asks
Join shown in table one.
Table one
The content that each known impurities in test sample are calculated by the reference substance external standard method with correction factor, by reference substance external standard method
The content of other single impurity in test solution is calculated, total impurities are the sum of each known impurities and other single impurity.
The method of the present invention can also carry out separation determination to doxercalciferol degradation impurity, and the preparation for sample to be tested of degrading is such as
Under：It takes doxercalciferol appropriate, sets in measuring bottle, use H2O2Dissolving, close plug take out, with organic solvent (example for 20 minutes in 80 DEG C of heating
Such as, methanol, ethyl alcohol or ether) dissolving, it is configured to the test solution of every lml 100 μ g containing doxercalciferol, takes 5 μ L injections high
Effect liquid phase chromatogram instrument completes the measurement of doxercalciferol alkaline degradation impurity according to above-mentioned chromatographic condition.Test solution is taken to inject
High performance liquid chromatograph records chromatogram, please specifically join shown in Fig. 1.In Fig. 1, the time that peak value appearance is detected in relation to substance 1 is
18.257 minutes, the time that doxercalciferol detects peak value appearance was 19.005 minutes, and the time that peak value occurs is detected in relation to substance 2
It it is 20.337 minutes, the time that peak value appearance is detected in relation to substance 3 is 20.939 minutes.
The series of detailed descriptions listed above only for the present invention feasible embodiment specifically
Bright, they are all without departing from equivalent implementations made by technical spirit of the present invention not to limit the scope of the invention
Or change should all be included in the protection scope of the present invention.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Profit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent requirements of the claims
Variation is included within the present invention.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiment being appreciated that.
Claims (5)
1. a kind of detection method measuring doxercalciferol and its impurities, the detection method use high performance liquid chromatography detection
Method, which is characterized in that the chromatographic condition of the detection method is：
Chromatographic column：Using octadecylsilane chemically bonded silica as stationary phase；
Mobile phase：The mixed solution of acid solution and organic solvent composition；
Flow velocity：1.5~2.0mL/min；
Detection wavelength：210nm；
Column temperature：40℃；
Sample size：5~10uL；
Sample concentration：3~5mg/mL；
The volume ratio of acid solution and organic solvent in the mixed solution with high performance liquid chromatography elution progress gradient
Lower, the volume ratio of acid solution and organic solvent in the mixed solution is：
The volume ratio of 0 min, acid solution and organic solvent is 30:70；
The volume ratio of 15min, acid solution and organic solvent is 28:72；
The volume ratio of 20min, acid solution and organic solvent is 15:85；
The volume ratio of 25min, acid solution and organic solvent is 15:85；
The volume ratio of 30min, acid solution and organic solvent is 0:100；
The organic solvent is selected from methanol or acetonitrile；
The chemical structural formula of the impurity is as follows：
。
2. detection method according to claim 1, which is characterized in that further include using organic solvent sample dissolution.
3. detection method according to claim 1, which is characterized in that the acid in the acid solution is selected from phosphoric acid, formic acid, second
One or two kinds of mixture with arbitrary proportion in acid, citric acid or trifluoroacetic acid.
4. detection method according to claim 1, which is characterized in that a concentration of 0.01 ~ 0.05vol% of the acid solution.
5. detection method according to claim 4, which is characterized in that a concentration of 0.05vol% of the acid solution.
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| CN108663442B (en) * | 2017-12-30 | 2021-03-30 | 南京海融制药有限公司 | Method for checking related substances of alfacalcidol tablet |
| CN111380976A (en) * | 2019-12-31 | 2020-07-07 | 卓和药业集团有限公司 | Analysis method of doxercalciferol injection |
| CN113237973B (en) * | 2021-05-07 | 2023-03-17 | 郑州泰丰制药有限公司 | Method for detecting content of impurities in eldecalcitol soft capsules |
| CN114839279A (en) * | 2022-02-21 | 2022-08-02 | 佛山市南海北沙制药有限公司 | Method for measuring content of impurities in vinyl ethyl ether |
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| WO2000043361A1 (en) * | 1999-01-20 | 2000-07-27 | Wisconsin Alumni Research Foundation | CRYSTALLINE 1α-HYDROXYVITAMIN D2 AND METHOD OF PURIFICATION THEREOF |
| CN101863809A (en) * | 2010-05-12 | 2010-10-20 | 重庆泰濠制药有限公司 | Method for purifying doxercalciferol |
| WO2011120162A1 (en) * | 2010-03-30 | 2011-10-06 | Alphora Research Inc. | Stabilized doxercalciferol and process for manufacturing the same |
| CN105237452A (en) * | 2015-11-01 | 2016-01-13 | 南京海融制药有限公司 | Novel crystalline form of doxercalciferol and preparation method for novel crystalline form |
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2016
- 2016-11-29 CN CN201611078612.8A patent/CN106770849B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000043361A1 (en) * | 1999-01-20 | 2000-07-27 | Wisconsin Alumni Research Foundation | CRYSTALLINE 1α-HYDROXYVITAMIN D2 AND METHOD OF PURIFICATION THEREOF |
| WO2011120162A1 (en) * | 2010-03-30 | 2011-10-06 | Alphora Research Inc. | Stabilized doxercalciferol and process for manufacturing the same |
| CN101863809A (en) * | 2010-05-12 | 2010-10-20 | 重庆泰濠制药有限公司 | Method for purifying doxercalciferol |
| CN105237452A (en) * | 2015-11-01 | 2016-01-13 | 南京海融制药有限公司 | Novel crystalline form of doxercalciferol and preparation method for novel crystalline form |
Non-Patent Citations (2)
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|---|
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| HPLC测定度骨化醇原料药的含量;李靖 等;《分析试验室》;20091130;第28卷;321-322 * |
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Effective date of registration: 20191227 Address after: 214000 no.219, Furong Middle Fourth Road, Xishan Economic and Technological Development Zone, Wuxi City, Jiangsu Province Patentee after: Zhuohe Pharmaceutical Group Co.,Ltd. Address before: 214000 No.2, Rongyang 1st Road, Xishan District, Wuxi City, Jiangsu Province Patentee before: Wuxi Fortune Pharmaceutical Co.,Ltd. |
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Address after: 219 Furong Zhongsi Road, Xishan Economic and Technological Development Zone, Wuxi City, Jiangsu Province, 214000 Patentee after: Zhuohe Pharmaceutical Group Co.,Ltd. Address before: 219 Furong Zhongsi Road, Xishan Economic and Technological Development Zone, Wuxi City, Jiangsu Province, 214000 Patentee before: Zhuohe Pharmaceutical Group Co.,Ltd. |