CN106525995A - Method for separating and measuring eplerenone and relevant substances by liquid chromatography - Google Patents
Method for separating and measuring eplerenone and relevant substances by liquid chromatography Download PDFInfo
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- CN106525995A CN106525995A CN201610836176.XA CN201610836176A CN106525995A CN 106525995 A CN106525995 A CN 106525995A CN 201610836176 A CN201610836176 A CN 201610836176A CN 106525995 A CN106525995 A CN 106525995A
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Abstract
The invention belongs to the field of analytical chemistry. The invention discloses a method for separating and measuring eplerenone and relevant substances by liquid chromatography. According to the method, chromatographic column with octadecylsilane bonded silica gel as the filling material is adopted, and a certain proportion of buffer salt solution-organic phase is used as a mobile phase so as to quantitatively measure contents of eplerenone and relevant substances. Then, quality of eplerenone is effectively controlled, and quality controllability of eplerenone is realized. The method of the invention has strong specialization and high accuracy and is simple to operate.
Description
Technical field
The invention belongs to analytical chemistry field, and in particular to liquid chromatography for separating and determining eplerenone and its relevant material
Method.
Background technology
Eplerenone is a kind of selective aldosterone receptor antagonists medicine, for treating hypertension and other angiocardiopathies.
The primary of not good low renin level is acted on to angiotensin converting enzyme inhibitor and angiotensin-ii receptor depressant
Hyperpietic, eplerenone have good antihypertensive effect.Further, it is also possible to significantly mitigate the ultrafiltration of glomerulus, so as to subtract
The albuminuria of light hyperpietic, and this has obvious Renoprotective Effect for the hyperpietic of complication with diabetes.According to
The chemical entitled 9,11 α-Epoxy-7 α of Puli's ketone-(methoxycarbonyl) -3-oxo-17 α-pregn-4-ene-21,17-
Carbolactone, molecular formula are C24H30O6.The structural formula of eplerenone is：
。
In the production process of the compound, there are several materials affect the purity and matter of medicine due to removing not exclusively
Amount, these materials are the relevant material in Control of drug quality, for eplerenone quality of production major control it is relevant
Material has 10, is 3-oxo-17 α-pregn-4-ene-7 α respectively, and 9:21,17-dicarbolactone（Relevant material 1）,
11α,12α-epoxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone
（Relevant material 2）, 7 α-(methoxycarbonyl) -3-oxo-17 α-pregna-4,9 (11)-diene-21,17-
Carbolactone (relevant materials 3）,（2’’R）-9,11α-epoxy-3,5’-dioxo-4’,5’-dihydro-3’H-spiro
[androst-4-ene-17,2’-furan]-7α-carboxylic acid（Relevant material 4）, 9,11 α-epoxy-7 β-
(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone（Relevant material 5）, 9,11 α-
epoxy-17-hydroxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21-carboxylic
sodium salt（Relevant material 6）, 7 α-(methoxycarbonyl) -3-oxo-17 α-pregn-4-ene-21,17-
carbolactone（Relevant material 7）, pregna-4-ene-7,21-dicarboxylic acid, 11,17-dihydroxy-
3-oxo,g-lactone,methyl ester,(7α,11α,17α)-（Relevant material 8）, (2 ' R, 7R, 10R, 11R, 13S)-
methyl 10,13-dimethyl-3,5’-dioxo-11-(tosyloxy)-1,2,3,4’,5’,6,7,8,9,10,11,12,
13,14,15,16-hexadecahydro-3’H-spiro[cyclopenta[α]phenanthrene-17,2’-furan]-7-
carboxylate（Relevant material 9）, 9,11-7 β -9,11 α-epoxy-7 α-(methoxycarbonyl) -3-oxo-17 α -
pregn-4-ene-21,17-carbolactone（Relevant material 10）, structural formula is respectively：
。
For the relevant material introduced in eplerenone art production process, need to carry out quality control in bulk drug,
Therefore, eplerenone and its separation about material is realized, is had important practical significance in terms of eplerenone quality control.
The content of the invention
It is an object of the invention to provide a kind of analyze eplerenone purity and separate its method about material, so as to
Separation and the measure of the associated material of eplerenone is realized, so as to ensure the purity of eplerenone, the matter of its finished product is realized
Amount control.
Use liquid chromatography analysis eplerenone purity of the present invention and its method about material is separated, be to adopt ten
Chromatographic column of the eight alkyl silane bonded silica gels for filler, with a certain proportion of buffer salt solution-organic phase as mobile phase.
Above-mentioned described chromatographic column with octadecylsilane chemically bonded silica as filler, chromatographic column be selected from Kromasil, Alltima and
The brands such as YMC.
Above-mentioned described organic phase is selected from following compound：Acetonitrile, propyl alcohol, isopropanol, tetrahydrofuran etc., preferably acetonitrile.
Above-mentioned described method, its mobile phase buffer salt solution-organic phase adopt gradient elution.
In above-mentioned described method, buffer salt solution is selected from phosphate, perchlorate, preferably phosphate.
Wherein the concentration of buffer salt solution is 0.02～0.1mol/L, and preferred concentration is 0.02mol/L.
Method of separating and assaying of the present invention, can be realized in accordance with the following methods：
1) eplerenone sample and its relevant material are taken appropriate, with acetonitrile or mobile phase sample dissolution, be configured to every 1mL containing according to
The sample solution of Puli 0.1～1.5mg of ketone；
2) flow rate of mobile phase is set for 0.5～1.5mL/min, flow rate of mobile phase is preferably 1.0mL/min, and Detection wavelength is 200
～250nm, best detection wavelength are 240nm, and column oven temperature is 10～40 DEG C, most preferably 40 DEG C of column oven temperature；
3) 10～50 μ L of sample solution 1) are taken, liquid chromatograph is injected, the separation for completing the associated material of eplerenone is surveyed
It is fixed.
Wherein：The model of high performance liquid chromatograph, has no special requirements, and the chromatograph that the present invention is adopted is for Shimadzu high performance liquid chromatography
Instrument：LC-20AT pumps, SPD-M20A detectors, SIL-20AC automatic samplers, CBM-20A controllers, CTO-10AS column ovens,
LC solution work stations；
Chromatographic column：C18（YMC, 150 × 4.6 mm, 3 μm）；
Mobile phase：A：0.02M ammonium dihydrogen phosphates, B：Acetonitrile；By being eluted with Gradient：
Flow velocity：1.0mL/min；
Detection wavelength：240nm；
Sampling volume：20μL；
Column temperature：40℃.
The present invention adopts C18（YMC, 150 × 4.6 mm, 3 μm）, eplerenone and its relevant material can be efficiently separated.This
Invention solves the problems, such as eplerenone and its separation determination about material, so as to reduce the generation of side reaction, improves original
Material medicine finished product yield and purity, it is ensured that eplerenone it is quality controllable.
Description of the drawings：
Eplerenone and its relevant material HPLC figures when Fig. 1 is embodiment 1；
Eplerenone HPLC figures when Fig. 2 is embodiment 1；
Blank solvent HPLC figure when Fig. 3 is embodiment 2；
Eplerenone and its relevant material HPLC figures when Fig. 4 is embodiment 2；
Eplerenone HPLC figures when Fig. 5 is embodiment 2.
Specific embodiment：
Following examples are used for further understanding the present invention, but are not limited to the scope of this enforcement.
Embodiment 1：
Instrument and condition
High performance liquid chromatograph：Shimadzu：LC-20AT, CBM-20A, SIL-20AC, SPD-M20A, CTO-10ASvp；
Chromatographic column：C18（YMC, 150 × 4.6 mm, 3 μm）；
Mobile phase：A：0.1% phosphoric acid water, B：Phosphoric acid-acetonitrile-methanol（0.1：40：60）；By being eluted with Gradient：
Flow velocity：1.0mL/min；
Detection wavelength：240nm；
Sampling volume：20μL；
Column temperature：30℃.
Experimental procedure：
Take eplerenone and its relevant material is appropriate, use acetonitrile sample dissolution respectively, be configured to containing eplerenone and its relevant thing
The sample solution of matter about 1.0mg/mL.Efficient liquid phase chromatographic analysis are carried out by above-mentioned condition, chromatogram is recorded.As a result see accompanying drawing 1 ~
In 2, Fig. 1, retention time is eplerenone for the chromatographic peak of 10.501min, and remaining chromatographic peak is each relevant material of eplerenone
Chromatographic peak；In Fig. 2, retention time is eplerenone for the chromatographic peak of 10.578min.
Embodiment 2：
Instrument and condition：
High performance liquid chromatograph：Shimadzu, LC-20AT, CBM-20A, SIL-20AC, SPD-M20A, CTO-10ASvp；
Chromatographic column：C18（YMC, 150 × 4.6 mm, 3 μm）；
Mobile phase：A：0.02M ammonium dihydrogen phosphates, B：Acetonitrile；By being eluted with Gradient：
Flow velocity：1.0mL/min；
Detection wavelength：240nm；
Sampling volume：20μL；
Column temperature：40℃.
Experimental procedure：
Take eplerenone and its relevant material is appropriate, use acetonitrile sample dissolution respectively, be configured to containing eplerenone and its relevant thing
The sample solution of matter about 1.0mg/mL；Acetonitrile is taken separately in right amount as blank solvent.High performance liquid chromatography point is carried out by above-mentioned condition
Analysis, records chromatogram.As a result see accompanying drawing 3 ~ 5, Fig. 3 is blank solvent chromatogram；Color of the retention time for 27.159min in Fig. 4
Spectral peak is eplerenone, remaining chromatographic peak be each chromatographic peak about material of eplerenone, as seen from the figure, eplerenone and
Its relevant material can reach baseline separation, meet the requirement of Chinese Pharmacopoeia；Chromatogram of the retention time for 27.096min in Fig. 5
Peak is eplerenone, it can be seen that the associated material of eplerenone can be kept completely separate under this condition.
The following items of above-mentioned eplerenone and its Related substance method are verified：
System suitability：
According to the chromatographic condition that above-described embodiment 2 determines, respectively with eplerenone and each mixed solution about material analyzing
Whether this chromatographic condition meets the requirements.Under the conditions of this, each meets the requirements about separating degree between material and main peak as seen from Figure 4,
Peak purity and single-point threshold values meet the requirements.
Sample introduction replica test：
By system suitability solution, the chromatographic condition as described in embodiment 2, repeat sample introduction 6 times, investigate the repeatability of method.By tying
Fruit understands that each is respectively less than 1.0% about the peak area RSD of material in continuous 6 subsystem applicability sample, and method repeatability is good
It is good.
。
Durability：
For the stability of further verification method, flow velocity, column temperature and chromatographic column brand these conditions are carried out accordingly by we
Fine setting, to investigate the durability of chromatographic condition.
As a result show, in the range of ± 0.2ml/min, in the range of ± 5 DEG C, peak type is not changed in change in flow for column temperature change,
Only retention time has corresponding reach and rear shifting.During the durability of chromatographic column is investigated, during Waters chromatogram column analysis, appearance is more early,
But the separating degree of each material is unsatisfactory for baseline separation.During Kromasil chromatogram column analysis, each material retention time and separating degree without
Significant change, each material peak purity and separating degree meet the requirements.
Test limit：
Take eplerenone appropriate, it is accurately weighed, acetonitrile sample dissolution is used, corresponding test liquid is configured to, then precision is measured for examination
Appropriate liquid, stepwise dilution are investigated by the chromatographic condition sample introduction of embodiment 2.
Eplerenone test limit data are as shown in the table：
。
Claims (11)
1. a kind of liquid chromatography for separating and determining eplerenone and its method about material, it is characterised in that：Octadecyl silicon
Chromatographic column of the alkane bonded silica gel for filler, with a certain proportion of buffer salt solution-organic phase as mobile phase.
2. method of separating and assaying according to claim 1, chromatographic column is selected from brands such as Kromasil, Alltima and YMC.
3. method of separating and assaying according to claim 1, the one kind or several of described organic phase in following compound
Kind：Acetonitrile, propyl alcohol, isopropanol, tetrahydrofuran etc..
4. method of separating and assaying according to claim 3, the preferred acetonitrile of described organic phase.
5. method of separating and assaying according to claim 1, described buffer salt solution are selected from following buffer salt：Phosphate,
Perchlorate etc..
6. method of separating and assaying according to claim 5, in described buffer salt solution, the concentration of contained buffer salt is preferred
0.02mol/L。
7. method of separating and assaying according to claim 5, buffer salt preferably phosphate in described buffer salt solution.
8. method of separating and assaying according to claim 1, it is characterised in that including following step：
1）Take eplerenone sample and its relevant material is appropriate, respectively with acetonitrile or mobile phase sample dissolution, be configured to every 1mL and contain
Eplerenone and its sample solution about 0.1～1.5mg of material；
2）Setting flow rate of mobile phase is 0.5～1.5mL/min, and Detection wavelength is 200～250nm, and column oven temperature is 10～40
℃；
3）Take 1）10～50 μ L of sample solution, inject liquid chromatograph, complete the separation survey of eplerenone and its relevant material
It is fixed.
9. method of separating and assaying according to claim 5, buffer salt preferably phosphoric acid ammonium dihydrogen.
10. method of separating and assaying according to claim 8, step 2）The preferred 1.0mL/min of described flow rate of mobile phase.
11. method of separating and assaying according to claim 8, step 2）The preferred 240nm of described Detection wavelength.
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| CN201610836176.XA CN106525995A (en) | 2016-09-21 | 2016-09-21 | Method for separating and measuring eplerenone and relevant substances by liquid chromatography |
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| CN201610836176.XA CN106525995A (en) | 2016-09-21 | 2016-09-21 | Method for separating and measuring eplerenone and relevant substances by liquid chromatography |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932505A (en) * | 2006-09-12 | 2007-03-21 | 石庆平 | Method for detecting eplerenone raw material or foreign matter and content in its preparation by effective liquid chromatography |
| CN101769905A (en) * | 2008-12-29 | 2010-07-07 | 北京德众万全药物技术开发有限公司 | Method utilizing HPLC (high performance liquid chromatography) to measure eplerenone cis-trans isomer |
| CN104844681A (en) * | 2014-02-13 | 2015-08-19 | 合肥久诺医药科技有限公司 | L-crystal form eplerenone refining method |
| CN105753930A (en) * | 2016-03-30 | 2016-07-13 | 北京万全德众医药生物技术有限公司 | Synthesizing method of eplerenone |
| CN105866298A (en) * | 2016-06-24 | 2016-08-17 | 合肥久诺医药科技有限公司 | High performance liquid chromatography analyzing method of eplerenone related substances |
-
2016
- 2016-09-21 CN CN201610836176.XA patent/CN106525995A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932505A (en) * | 2006-09-12 | 2007-03-21 | 石庆平 | Method for detecting eplerenone raw material or foreign matter and content in its preparation by effective liquid chromatography |
| CN101769905A (en) * | 2008-12-29 | 2010-07-07 | 北京德众万全药物技术开发有限公司 | Method utilizing HPLC (high performance liquid chromatography) to measure eplerenone cis-trans isomer |
| CN104844681A (en) * | 2014-02-13 | 2015-08-19 | 合肥久诺医药科技有限公司 | L-crystal form eplerenone refining method |
| CN105753930A (en) * | 2016-03-30 | 2016-07-13 | 北京万全德众医药生物技术有限公司 | Synthesizing method of eplerenone |
| CN105866298A (en) * | 2016-06-24 | 2016-08-17 | 合肥久诺医药科技有限公司 | High performance liquid chromatography analyzing method of eplerenone related substances |
Non-Patent Citations (1)
| Title |
|---|
| 赵海龙 等: "高效液相色谱法测定依普利酮的含量和有关物质", 《中国药学杂志》 * |
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Application publication date: 20170322 |