CN104062381A - Method for determining contents of diphosphonic acid compounds - Google Patents
Method for determining contents of diphosphonic acid compounds Download PDFInfo
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- CN104062381A CN104062381A CN201410330791.4A CN201410330791A CN104062381A CN 104062381 A CN104062381 A CN 104062381A CN 201410330791 A CN201410330791 A CN 201410330791A CN 104062381 A CN104062381 A CN 104062381A
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Abstract
The invention discloses a method for determining the contents of diphosphonic acid compounds. The method comprises the steps: determining a to-be-tested sample containing the diphosphonic acid compounds by using a high performance liquid chromatography to obtain a peak area of a main peak of the to-be-tested sample, wherein a used mobile phase is an anhydrous formic acid solution; regulating the pH value to be 3.4-3.6 by using triethylamine, wherein the volume ratio of the anhydrous formic acid to water is (0.18-0.22)/1000; and calculating the contents of the diphosphonic acid compounds in the to-be-tested sample by using an external standard method. The method is simple and easy to operate, convenient to operate, relatively wide in detection concentration range, relatively good in repeatability and relatively high in precision and accuracy.
Description
Technical field
The invention belongs to the instrumental analysis field of chemical pharmacy, be specifically related to a kind of content assaying method of bis-phosphonic acids compounds.
Background technology
Bisphosphonates is a kind of important treatment osteoporosis agents, its effective constituent is bis-phosphonic acids compounds, oral or intravenous administration, is used for the treatment of hypercalcinemia and ostalgia etc. that osteoporosis, scleromalacia disease, malignant metastatic tumor of bone cause.
Bisphosphonate class of drugs is broken the potent inhibitor of bone effect, wherein T
1/2: reach 12 years, it is very lasting to act on.Bis-phosphonic acids compounds can be adsorbed in the surface of the light base apatite of chief component of bone securely, suppresses its formation and dissolving, also can suppress the calcification of soft tissue and the absorption of bone.Developed up to now " 3 generation product ", rapidly, it suppresses, and bone absorbs and to increase bone amount evident in efficacy, and more easily tolerance in bisphosphonate class of drugs development, and toxic and side effect is little, is considered to the most promising at present, and bone is absorbed to one of the strongest inhibitor.
Bis-phosphonic acids compounds is because its compound property is special, in the time using high performance liquid chromatography to measure, is normally usedly difficult to carry out assay taking C18 post, UV-detector as basic reverse-phase chromatography condition.The present invention aim to provide one efficiently, assay method efficiently, Accurate Determining bis-phosphonic acids compounds content.
Summary of the invention
The content assaying method that the invention provides a kind of bis-phosphonic acids compounds, the method is simple, convenient operation.
Technical solution of the present invention is as follows:
A kind of content assaying method of bis-phosphonic acids compounds, its special character is, described assay method is the testing sample containing bis-phosphonic acids compounds with high effective liquid chromatography for measuring, obtain the peak area of testing sample main peak, wherein mobile phase used is anhydrous formic acid aqueous solution, triethylamine is adjusted pH value to 3.4～3.6, and the volume ratio of anhydrous formic acid and water is 0.18～0.22/1000, calculates the content of bis-phosphonic acids compounds in testing sample by external standard method.
Based on technique scheme, the present invention also makes following optimization and restriction:
In this assay method, the chromatographic column of selecting is anion exchange resins chromatographic column, and detecting device is differential refraction detector; The packing material size of chromatographic column is 7 μ m, and filler contains quaternary ammonium salt group, and filler is the resin taking methacrylate as lattice.
In this assay method, the concentration of the sample solution of preparation and standard solution preparation is 1.6～2.4mg/ml, and solvent is pure water.
The flow velocity of above-mentioned mobile phase is 1.8-2.2ml/min, and sample size is 100 μ l.
The column temperature of above-mentioned chromatographic column is 32-38 DEG C.
The minute of said determination method is the twice of main peak retention time, and minute starts timing by the high performance liquid chromatograph sample introduction moment.
The volume ratio of above-mentioned anhydrous formic acid and water is 0.2/1000, and the pH value of anhydrous formic acid aqueous solution is 3.5, and the concentration of sample solution and standard solution preparation is 2.0mg/ml, and the flow velocity of mobile phase is 2.0ml/min, and the column temperature of chromatographic column is 35 DEG C.
Advantage of the present invention is:
1, there is higher good selectivity, sensitivity and accuracy, and easy and simple to handle, measure fast, simple.
2, the mobile phase in the present invention also can be for the related assays of liquid chromatograph mass spectrography (LC-MS), to measuring containing content and the corresponding molecular structure of impurity in the sample of bis-phosphonic acids compounds.
3, the mobile phase in the present invention, does mobile phase with volatilizable salt, reduces the obstruction of salt for liquid chromatography pipeline.
4, the present invention has wider detectable concentration scope, also has good repeatability and higher preci-sion and accuracy.
Brief description of the drawings
Fig. 1: the high-efficient liquid phase chromatogram that obtains bis-phosphonic acids compounds Alendronate sodium according to method of the present invention.
Embodiment
The invention discloses a kind of content assaying method of bis-phosphonic acids compounds, the method is the testing sample containing bis-phosphonic acids compounds with high effective liquid chromatography for measuring, obtains the peak area of testing sample main peak,, select high performance liquid chromatograph as detecting instrument; Wherein mobile phase used is anhydrous formic acid aqueous solution, and triethylamine is adjusted pH value to 3.4～3.6, and the volume ratio of anhydrous formic acid and water is 0.18～0.22/1000, excellent 0.2/1000, with the content of bis-phosphonic acids compounds in external standard method calculating testing sample.Minute of the present invention is the twice of main peak retention time, and minute starts timing by the high performance liquid chromatograph sample introduction moment, and main peak retention time is appearance time.Wherein, the flow velocity of mobile phase is 1.8-2.2ml/min, preferably 2.0ml/min, and sample size is 100 μ l.The pure level of the general at least Analysis about Selection of anhydrous formic acid in the present invention and triethylamine.
The chromatographic column that the present invention selects is the anion exchange resins chromatographic column of 150 × 4.6mm, and detecting device is differential refraction detector; In chromatographic column, the particle diameter of filler is 7 μ m, and filler contains quaternary ammonium salt group, and filler is the resin taking methacrylate as lattice; The detecting device that high performance liquid chromatography is selected is differential refraction detector.The column temperature of chromatographic column is 32-38 DEG C, preferably 35 DEG C.
In the present invention, the concentration of the sample solution of preparation and standard solution preparation is 1.6～2.4mg/ml, and solvent is pure water, preferably selects the pure water of HPLC level.
The process of preparation sample solution is: precision takes bis-phosphonic acids compounds testing sample 50.0 ± 0.5mg, accurately weighed, puts in 25ml volumetric flask, is diluted to scale with pure water, shakes up, for subsequent use.
The process of preparing standard solution is: precision takes bis-phosphonic acids compounds standard items 50.0 ± 0.5mg and is placed in a 25ml volumetric flask, is diluted to scale with pure water, shakes up, for subsequent use.
External standard method computing formula:
In formula:
the peak area of main peak in need testing solution;
the peak area of main peak in standard solution;
WSTD: the sample weighting amount of standard solution Plays product, the mg of unit;
WSPL: the sample weighting amount of test sample in need testing solution, the mg of unit;
P: the content (as is) of standard items, the mg/mg of unit.
According to following chromatographic condition, repeatability of the present invention, precision, accuracy and sensing range (upper limit, lower limit) are verified:
[chromatographic condition]
Chromatograph: high performance liquid chromatograph;
Chromatographic column: 150 × 4.6mm, packing material size is 7 μ m, anion exchange resins, the resin taking methacrylate as lattice that filler contains quaternary ammonium salt group;
Mobile phase: anhydrous formic acid aqueous solution, the volume ratio of anhydrous formic acid and water is 0.2/1000, with triethylamine adjust pH be 3.5;
Detecting device: differential refraction detector;
Flow velocity: 2.0ml/min;
Sample size: 100 μ l;
Column temperature: 35 DEG C;
Minute: be the twice of main peak retention time (appearance time).
Choose a certain sample that contains Alendronate sodium as testing sample, it is 99.98% Alendronate sodium EP standard items that standard items are chosen massfraction, take 6 parts of testing samples and 1 part of standard items, take and the results are shown in Table one, according to the present invention, the standard items that take and 6 parts of testing samples are all formulated as to the solution that concentration is 2.0mg/ml, and respectively every part of solution of preparation is measured, obtain every part of corresponding peak area of solution main peak, the massfraction that calculates 6 parts of testing samples according to external standard method computing formula, result of calculation is in Table
Two:
Table one
Table two
Choose as the same standard items in table one, take respectively 7 parts of standard items, will be wherein 6 parts as testing sample, residue portion is still standard items, take result as table three, testing sample and standard items are all formulated as to the solution that concentration is 2.0mg/ml, respectively every part of solution of preparation is measured, obtain every part of corresponding peak area of solution main peak, calculate the massfraction of 6 parts of testing samples according to external standard method computing formula, according to the massfraction of 6 parts of testing samples that calculate, calculate the recovery of 6 parts of testing samples according to recovery computing formula again
Recovery computing formula:
Recovery result of calculation is in table four.
Table three
Table four
Choose as the same standard items in table one, take respectively 7 parts of standard items, will be wherein 6 parts as testing sample, residue portion is still standard items, take result as table five, testing sample is formulated as to the solution that concentration is 1.6mg/ml, standard items are formulated as the solution that concentration is 2.0mg/ml, respectively every part of solution of preparation is measured, obtain every part of corresponding peak area of solution main peak, calculate the massfraction of 6 parts of testing samples according to external standard method computing formula, again according to the massfraction of 6 parts of testing samples that calculate, calculate the recovery of 6 parts of testing samples according to recovery computing formula, recovery result of calculation is in table six.
Table five
Table six
Choose as the same standard items in table one, take respectively 7 parts of standard items, will be wherein 6 parts as testing sample, residue portion is still standard items, take result as table seven, testing sample is formulated as to the solution that concentration is 2.4mg/ml, standard items are formulated as the solution that concentration is 2.0mg/ml, respectively every part of solution of preparation is measured, obtain every part of corresponding peak area of solution main peak, calculate the massfraction of 6 parts of testing samples according to external standard method computing formula, again according to the massfraction of 6 parts of testing samples that calculate, calculate the recovery of 6 parts of testing samples according to recovery computing formula, recovery result of calculation is in table eight.
Table seven
Table eight
According to table two, table four, table six and table eight, illustrate that this method has good repeatability and higher precision; Illustrate that according to table four, table six and table eight the present invention has higher accuracy; According to table two, table six and table eight, illustrate that the present invention can be used for measuring the solution that concentration is 1.6～2.4mg/ml, there is wider detectable concentration scope.Therefore the invention reside in while measuring the solution that concentration is 1.6～2.4mg/ml, there is good repeatability and higher preci-sion and accuracy.
Claims (7)
1. the content assaying method of a bis-phosphonic acids compounds, it is characterized in that, described assay method is the testing sample containing bis-phosphonic acids compounds with high effective liquid chromatography for measuring, obtain the peak area of testing sample main peak, wherein mobile phase used is anhydrous formic acid aqueous solution, triethylamine is adjusted pH value to 3.4～3.6, and the volume ratio of anhydrous formic acid and water is 0.18～0.22/1000, calculates the content of bis-phosphonic acids compounds in testing sample by external standard method.
2. the content assaying method of bis-phosphonic acids compounds according to claim 1, is characterized in that, in this assay method, the chromatographic column of selecting is anion exchange resins chromatographic column, and detecting device is differential refraction detector; The packing material size of chromatographic column is 7 μ m, and filler contains quaternary ammonium salt group, and filler is the resin taking methacrylate as lattice.
3. the content assaying method of bis-phosphonic acids compounds according to claim 2, is characterized in that, in this assay method, the concentration of the sample solution of preparation and standard solution preparation is 1.6～2.4mg/ml, and solvent is pure water.
4. the content assaying method of bis-phosphonic acids compounds according to claim 3, is characterized in that, the flow velocity of described mobile phase is 1.8-2.2ml/min, and sample size is 100 μ l.
5. the content assaying method of bis-phosphonic acids compounds according to claim 4, is characterized in that, the column temperature of described chromatographic column is 32-38 DEG C.
6. the content assaying method of bis-phosphonic acids compounds according to claim 5, is characterized in that,
The minute of described assay method is the twice of main peak retention time, and minute starts timing by the high performance liquid chromatograph sample introduction moment.
7. the content assaying method of bis-phosphonic acids compounds according to claim 6, it is characterized in that, the volume ratio of described anhydrous formic acid and water is 0.2/1000, the pH value of anhydrous formic acid aqueous solution is 3.5, the concentration of sample solution and standard solution preparation is 2.0mg/ml, the flow velocity of mobile phase is 2.0ml/min, and the column temperature of chromatographic column is 35 DEG C.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090114741A (en) * | 2008-04-30 | 2009-11-04 | 주식회사 아이바이오팜 | Method of analyzing bisphophonate compound using derivative reaction |
| US20120193528A1 (en) * | 2007-03-07 | 2012-08-02 | Sanofi-Aventis U.S. Llc | Quantitative determination of risedronate in urine by spe-lc-ms-ms |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120193528A1 (en) * | 2007-03-07 | 2012-08-02 | Sanofi-Aventis U.S. Llc | Quantitative determination of risedronate in urine by spe-lc-ms-ms |
| KR20090114741A (en) * | 2008-04-30 | 2009-11-04 | 주식회사 아이바이오팜 | Method of analyzing bisphophonate compound using derivative reaction |
Non-Patent Citations (5)
| Title |
|---|
| BRITISH PHARMACOPOEIA COMMISSION SECRETARIAT: "Sodium Alendronate", 《BRITISH PHARMACOPOEIA 2010》 * |
| YIENG-HAU R. HAN ET AL.: "Determination of alendronate sodium by ion chromatography with refractive index detection", 《JOURNAL OF CHROMATOGRAPHY A》, vol. 719, 31 December 1996 (1996-12-31) * |
| 唐克慧等: "离子交换HPLC 法测定唑来膦酸的含量及有关物质", 《中国抗生素杂志》, vol. 30, no. 2, 28 February 2005 (2005-02-28), pages 88 - 90 * |
| 谢智远等: "高效液相色谱法测定阿仑膦酸钠片的含量", 《中国药房》, vol. 12, no. 9, 31 December 2001 (2001-12-31) * |
| 门磊等: "高效液相色谱法测定尿液中阿仑膦酸钠的浓度", 《中国药学杂志》, vol. 45, no. 19, 31 October 2010 (2010-10-31), pages 1492 - 1495 * |
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Application publication date: 20140924 |