CN104345098B - A kind of method measuring antioxidant content in tablet of tibolone - Google Patents
A kind of method measuring antioxidant content in tablet of tibolone Download PDFInfo
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- CN104345098B CN104345098B CN201310335362.1A CN201310335362A CN104345098B CN 104345098 B CN104345098 B CN 104345098B CN 201310335362 A CN201310335362 A CN 201310335362A CN 104345098 B CN104345098 B CN 104345098B
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Abstract
The invention provides a kind of method measuring antioxidant content in tablet of tibolone, concrete is a kind of method by antioxidant composition 6-palmityl-L-AA content in rp-hplc determination tablet of tibolone.This method is easy and simple to handle, and specificity is better, can avoid the interference that the composition of the auxiliary material in tablet and Tibolone measures for antioxidant content, and sample is stablized, and accuracy is high, and applicability is better.
Description
Technical field
The present invention relates to the method for tablet of tibolone Control of Impurities, be specifically related to a kind of method measuring 6-palmityl-L-AA content in tablet of tibolone.
Background technology
Tibolone (tibolone) is a kind of steroid hormone class medicine of Prof. Du Yucang, and chemical name is 17-hydroxyl-7 Alpha-Methyl-19-demethyl-17 α-pregnant steroid-5 (10)-alkene-20-alkynes-3-ketone.This clinical drug is widely used in prevent and treat cardiovascular and cerebrovascular disease, osteoporosis, alleviating menopausal depressive symptom, reconciles lipid metaboli, treatment climacteric or menopausal women disorder etc. are menopausal women hormone replacement therapy medicines ideal at present.
Tibolone is easily oxidized and produce the impurity relevant with oxidation, and use vitamin E as antioxidant in traditional preparation process thereof, vitamin E is oily, needs heating for dissolving in preparation, causes antioxidant effect greatly to reduce; Find in preparation research process, add 6-palmityl-L-AA, for active constituents of medicine, there is better stabilization.The chemical name of this antioxidant composition is (S)-2-[(R)-3,4-dihydroxy-5-oxo-2,5-dihydro-2-furyl]-2-hydroxyethyl Palmitate, and chemical structural formula is:
Adding of 6-palmityl-L-AA effectively can reduce impurity content relevant with oxidation in Tibolone sheet, when the content of 6-palmityl-L-AA reduces, its antioxidation weakens, can corresponding increase with the relevant impurity of oxidation, affects security and the validity of medicine; And as a kind of auxiliary material of medicine, the content of antioxidant also needs clear and definite and controls, in addition 6-palmityl-L-AA itself also easy oxidized generation degraded thus affect its content, therefore needs the quality of content to tablet of tibolone by measuring 6-palmityl-L-AA to control.
The regulation detecting antioxidant 6-palmityl-L-AA content is there is no in current existing tablet of tibolone quality standard.Measuring iodimetry that 6-palmityl-L-AA raw material commonly uses due to interference problem is not suitable for the assay of this composition in tablet of tibolone.
For improving the quality of tablet of tibolone, the oxidation impurities solved in tablet of tibolone controls relevant problem, be necessary the assay method proposing 6-palmityl-L-AA antioxidant content in a kind of tablet of tibolone, can measure this antioxidant content in the quality research of product and stability study.
Summary of the invention
An object of the present invention is to provide a kind of method measuring 6-palmityl-L-AA content in tablet of tibolone.
Two of object of the present invention is the oxidation impurities control method providing a kind of tablet of tibolone.
Technical scheme of the present invention is:
Utilize high performance liquid chromatography, with 6-palmityl-L-AA for reference substance, take arabo-ascorbic acid as stabilizing agent, measure as the content of 6-palmityl-L-AA in the described tablet of tibolone of test sample, calculated the content of 6-palmityl-L-AA in tablet of tibolone by external standard method.
This method mobile phase used comprises inorganic phase A and organic phase B, and wherein A is phosphoric acid-aqueous systems, and B is acetonitrile-methanol system.
The volume ratio V of A and B in the mobile phase that this method is used
a/ V
b=15/85 ~ 5/95, preferably 5/95; The volume ratio V of phosphoric acid-water
phosphoric acid/ V
water=1/99; The volume ratio V of acetonitrile-methanol
acetonitrile/ V
methyl alcohol=45/55 ~ 55/45, preferably 50/50.Flow rate of mobile phase is 0.8mL/min1mL/min, preferred 1mL/min.Chromatographic column is C18 reverse-phase chromatographic column, and chromatographic column can adopt the specification of 250 × 1.6mm5 μm or 150 × 4.6mm5 μm, such as InertsilODS-3250 × 1.6mm5 μm, DiamonsilC18150 × 4.6mm5 μm or AgilentC18150 × 4.6mm5 μm.Column temperature is set as 25 ~ 35 DEG C, and preferred column temperature is 30 DEG C.UV-detector determined wavelength is 243nm.
The preparation of following solution is also comprised in this method:
I. the preparation of Extraction solvent
Get arabo-ascorbic acid and be placed in volumetric flask in right amount, dissolve with methyl alcohol and be settled to scale, be prepared into arabo-ascorbic acid methanol solution, be preferably mixed with the arabo-ascorbic acid methanol solution that mass concentration is 1mg/mL.
II. the preparation of need testing solution
Get Tibolone sheet and be placed in volumetric flask in right amount, add a small amount of water, jolting, after making tablet disintegrates, adds appropriate Extraction solvent, put after ultrasonic to room temperature, add Extraction solvent and be settled to scale, shake up, filter, get subsequent filtrate and be prepared into need testing solution, be preferably prepared into the need testing solution that mass concentration is 0.02mg/mL.
III. the preparation of reference substance solution
Get 6-palmityl-L-AA reference substance appropriate, be placed in volumetric flask, add appropriate Extraction solvent, put after ultrasonic to room temperature, add Extraction solvent and be settled to scale, shake up, be prepared into reference substance solution, be preferably prepared into the reference substance solution that mass concentration is 0.02mg/mL.
This method also comprises the following step calculating content:
Respectively accurately draw reference substance solution and each 10 μ L of need testing solution, injection liquid chromatography, record chromatogram, by external standard method with calculated by peak area content and get final product.
Content (mg/ the sheet)=A of 6-palmityl-L-AA in tablet of tibolone
x× C
s/ A
s× D/ sheet number
In formula: A
x: the peak area of 6-palmityl-L-AA in need testing solution;
C
s: the actual concentrations of 6-palmityl-L-AA in reference substance solution;
A
s: the peak area of 6-palmityl-L-AA in reference substance solution;
D: extension rate
Mobile phase of the present invention adopts known method to prepare.This method tablet of tibolone used obtains by buying.
Beneficial effect of the present invention is as follows:
1, this method adopts arabo-ascorbic acid as the stabilizing agent in Sample Preparation Procedure, and overcome the shortcoming that 6-palmityl-L-AA is easily degraded, in mensuration process, sample stability is high, favorable reproducibility.
2, the composition that this method avoids auxiliary material and Tibolone, for the interference phenomenon of 6-palmityl-L-AA assay, has better specificity.
3, this method is easy and simple to handle, and accuracy is good, and durability is strong.
Accompanying drawing explanation
1-Fig. 1 is the HPLC collection of illustrative plates of reference substance solution in embodiment 1
2-Fig. 2 is the HPLC collection of illustrative plates of need testing solution in embodiment 1
3-Fig. 3 is the HPLC collection of illustrative plates of Extraction solvent arabo-ascorbic acid methanol solution in embodiment 1
4-Fig. 4 is the HPLC collection of illustrative plates of need testing solution in embodiment 2 (not adding 6-palmityl-L-AA)
Embodiment
Embodiment 1:
(1) solution preparation
I. the preparation of Extraction solvent
Take arabo-ascorbic acid 0.5g and be placed in 500mL volumetric flask, dissolve with methyl alcohol and be settled to scale, be prepared into the arabo-ascorbic acid methanol solution that mass concentration is 1mg/mL.
II. the preparation of need testing solution
Get Tibolone sheet 10 (specification is 2.5mg), be placed in 100mL volumetric flask, add 10mL water, jolting, after making tablet disintegrates, add appropriate Extraction solvent, ultrasonic 10min, put to room temperature, add Extraction solvent and be settled to scale, shake up, filter, get subsequent filtrate and be diluted to the need testing solution that mass concentration is 0.02mg/mL.
III. the preparation of reference substance solution
Get 6-palmityl-L-AA reference substance 10mg, be placed in 50mL volumetric flask, add appropriate Extraction solvent, ultrasonic 10min, put to room temperature, add Extraction solvent and be settled to scale, shake up, obtain 6-palmityl-L-AA storing solution, precision measures this storing solution 1mL, is placed in 10mL measuring bottle, adds Extraction solvent and is settled to scale, shake up, obtained quality solubility is the reference substance solution of 0.02mg/mL.
(2) operation condition of chromatogram is set:
I. mobile phase: the volume ratio V of inorganic phase A/ organic phase B
a/ V
b=5/95, wherein, A is the volume ratio V of phosphoric acid-aqueous systems, phosphoric acid/water
phosphoric acid/ V
water=1/99; B is the volume ratio V of acetonitrile-methanol system, acetonitrile/methanol
acetonitrile/ V
methyl alcohol=50/50
II. flow rate of mobile phase: 1mL/min
III. UV-detector determined wavelength: 243nm
IV. chromatographic column: InertsilODS-3C18 reverse-phase chromatographic column, 250 × 1.6mm, 5 μm
V. column temperature: 30 DEG C
(3) assay
Respectively accurately draw reference substance solution and each 10 μ L of need testing solution, injection liquid chromatography, record chromatogram (result is as accompanying drawing 1, accompanying drawing 2), by external standard method with calculated by peak area content and get final product.
Content (mg/ the sheet)=A of 6-palmityl-L-AA in tablet of tibolone
x× C
s/ A
s× 100/10
In formula: A
x: the peak area of 6-palmityl-L-AA in need testing solution;
C
s: the actual concentrations of 6-palmityl-L-AA in reference substance solution;
A
s: the peak area of 6-palmityl-L-AA in reference substance solution;
Embodiment 2:
The detection method of embodiment 1 is investigated.
(1) specificity
1,6-palmityl-L-AA reference substance solution: adopt the 6-palmityl-L-AA reference substance solution prepared in embodiment 1.
2, blank Extraction solvent: the Extraction solvent arabo-ascorbic acid methanol solution prepared in embodiment 1 is as blank Extraction solvent.
3, need testing solution (not adding 6-palmityl-L-AA): the Tibolone sheet 10 (specification is 2.5mg) getting the homemade 6-of interpolation palmityl-L-AA, adopt the method preparing need testing solution in embodiment 1, obtain the solution not investigating contrast containing 6-palmityl-L-AA for specificity.
Adopt the chromatographic condition of embodiment 1, respectively each 10 μ L of the above-mentioned three kinds of solution of accurate absorption, injection liquid chromatography, record chromatogram, the results are shown in accompanying drawing 1,3, shown in 4.
(2) stability of solution
1, need testing solution: get Tibolone sheet 10 (specification is 2.5mg), be placed in 100mL volumetric flask, add 10mL water, jolting, after making tablet disintegrates, add appropriate Extraction solvent (the 1mg/mL arabo-ascorbic acid methanol solution obtained according to the method for embodiment 1), ultrasonic 10min, puts to room temperature, add Extraction solvent and be settled to scale, shake up, filter, get subsequent filtrate as need testing solution.
2, the contrast solution of stabilizing agent is not added: get Tibolone sheet 10 (specification is 2.5mg), be placed in 100mL volumetric flask, add 10mL water, jolting, after making tablet disintegrates, add proper amount of methanol solution, ultrasonic 10min, put to room temperature, add methanol solution and be settled to scale, shake up, filter, get subsequent filtrate as the contrast solution not adding stabilizing agent.
Under identical condition, precision measures each 10 μ L of above-mentioned two kinds of solution respectively, injection liquid chromatography, and obtain the peak area situation of change in time of 6-palmityl-L-AA according to chromatogram, result is as shown in table 1:
Table 1: the study on the stability of solution
(3) linear
Get ascorbyl palmitate reference substance 10mg, accurately weighed, put in 100mL volumetric flask, add Extraction solvent dissolve and be diluted to scale, shake up, obtained mass concentration is the ascorbyl palmitate solution of 0.1mg/mL, pipetting this solution appropriate respectively according to following table 2 puts in suitable measuring bottle, scale is diluted to Extraction solvent (the 1mg/mL arabo-ascorbic acid methanol solution obtained according to the method for embodiment 1), shake up, namely obtain variable concentrations linear solvent (50% ~ 150%).Map to concentration with peak area, drawing standard curve, fitting linear equation is y=1245.6x-3.8988, R
2=0.9998; Concrete outcome is as shown in table 2:
Table 2: linearly investigate
(4) accuracy
Precision takes Tibolone sheet and (does not add 6-palmityl-L-AA respectively, specification is 2.5mg) 2.5 slices, 5, 2.5 are placed in 25ml respectively, 50ml, in 25ml measuring bottle, add water jolting, after making tablet disintegrates, precision adds 6-palmityl-L-AA storing solution 2.0ml obtained in embodiment 1 successively again, 5.0ml, 3.0ml, add appropriate Extraction solvent (the 1mg/mL arabo-ascorbic acid methanol solution obtained according to the method for embodiment 1) respectively, ultrasonic 10min makes dissolving, place room temperature, add Extraction solvent and be settled to scale, shake up, precision draws each 10 μ L of solution prepared respectively, injection liquid chromatography, record chromatogram, calculate corresponding content, measurement result is as shown in table 3:
Table 3: method accuracy is investigated
Claims (7)
1. one kind measures the method for the 6-palmityl-L-AA content in tablet of tibolone, it is characterized in that with 6-palmityl-L-AA for reference substance, take arabo-ascorbic acid as stabilizing agent, adopt reversed-phased high performace liquid chromatographic to measure the content of 6-palmityl-L-AA in described tablet of tibolone, calculate content by external standard method; Wherein high performance liquid chromatography chromatographic column used is C18 reverse-phase chromatographic column, and mobile phase comprises inorganic phase A and organic phase B, and wherein A is phosphoric acid-aqueous systems, and B is the volume ratio V of acetonitrile-methanol system, A and B
a/ V
b=15/85 ~ 5/95, the volume ratio V of phosphoric acid-water
phosphoric acid/ V
water=1/99, the volume ratio V of acetonitrile-methanol
acetonitrile/ V
methyl alcohol=45/55 ~ 55/45.
2. method according to claim 1, is characterized in that V in mobile phase
a/ V
b=5/95, V
acetonitrile/ V
methyl alcohol=50/50.
3. method according to claim 2, it is characterized in that flow rate of mobile phase is 0.8mL/min ~ 1mL/min, UV-detector determined wavelength is 243nm, and chromatographic column is C18 reverse-phase chromatographic column, and column temperature is 25 ~ 35 DEG C.
4. method according to claim 3, it is characterized in that flow rate of mobile phase is 1mL/min, column temperature is 30 DEG C, and chromatographic column is C18250 × 1.6mm5 μm or 150 × 4.6mm5 μm.
5. method according to claim 4, is characterized in that chromatographic column is InertsilODS-3250 × 1.6mm5 μm.
6. the method according to Claims 1 to 5 any one claim, is characterized in that mensuration process comprises the preparation of following solution:
I. the preparation of Extraction solvent
Get arabo-ascorbic acid and be placed in volumetric flask in right amount, dissolve with methyl alcohol and be settled to scale, be prepared into the arabo-ascorbic acid methanol solution that mass concentration is 1mg/mL;
II. the preparation of need testing solution
Get Tibolone sheet and be placed in volumetric flask in right amount, add a small amount of water, jolting, after making tablet disintegrates, add appropriate Extraction solvent, put to room temperature after ultrasonic, add Extraction solvent and be settled to scale, shake up, filter, get subsequent filtrate and be prepared into the need testing solution that mass concentration is 0.02mg/mL;
III. the preparation of reference substance solution
Get 6-palmityl-L-AA reference substance appropriate, be placed in volumetric flask, add appropriate Extraction solvent, put to room temperature after ultrasonic, add Extraction solvent and be settled to scale, shake up, be prepared into the reference substance solution that mass concentration is 0.02mg/mL.
7. method according to claim 6, is characterized in that adopting and carries out cubage with the following method:
Content=the A of 6-palmityl-L-AA in tablet of tibolone
x× C
s/ A
s× D/ sheet number, wherein the unit of content is mg/ sheet, in formula: A
x: the peak area of 6-palmityl-L-AA in need testing solution;
C
s: the actual concentrations of 6-palmityl-L-AA in reference substance solution;
A
s: the peak area of 6-palmityl-L-AA in reference substance solution;
D: dilution volume.
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Citations (3)
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|---|---|---|---|---|
| CN1253503A (en) * | 1997-04-22 | 2000-05-17 | 阿克佐诺贝尔公司 | Stabilized tibolone compositions |
| CN1713898A (en) * | 2002-11-20 | 2005-12-28 | 阿克佐诺贝尔公司 | Pharmaceutical tablets containing tibolone and a coating |
| CN102749394A (en) * | 2012-06-06 | 2012-10-24 | 南京农业大学 | Separation and measurement method for reduction-type ascorbic acid and erythorbic acid in fruit and vegetable tissues and related products |
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2013
- 2013-08-05 CN CN201310335362.1A patent/CN104345098B/en active Active
Patent Citations (3)
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|---|---|---|---|---|
| CN1253503A (en) * | 1997-04-22 | 2000-05-17 | 阿克佐诺贝尔公司 | Stabilized tibolone compositions |
| CN1713898A (en) * | 2002-11-20 | 2005-12-28 | 阿克佐诺贝尔公司 | Pharmaceutical tablets containing tibolone and a coating |
| CN102749394A (en) * | 2012-06-06 | 2012-10-24 | 南京农业大学 | Separation and measurement method for reduction-type ascorbic acid and erythorbic acid in fruit and vegetable tissues and related products |
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| Title |
|---|
| L-抗坏血酸棕榈酸酯的研究进展;章立群 等;《郑州粮食学院学报》;19980331;第19卷;89-95,99 * |
| RP-HPLC法测定替勃龙原料药有关物质及含量;赵燕燕 等;《药物分析杂志》;20120630;第32卷(第6期);994-997 * |
| Simultaneous Determination of Ascorbyl Palmitate and Nine Phenolic Antioxidants in Vegetable Oils and Edible Fats by HPLC;Christian Perrin et al.;《JAOCS》;20030228;第80卷(第2期);115-118 * |
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