CN101504393B - HPLC measuring method for d-biotin and its impurity content - Google Patents
HPLC measuring method for d-biotin and its impurity content Download PDFInfo
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- CN101504393B CN101504393B CN2008101621386A CN200810162138A CN101504393B CN 101504393 B CN101504393 B CN 101504393B CN 2008101621386 A CN2008101621386 A CN 2008101621386A CN 200810162138 A CN200810162138 A CN 200810162138A CN 101504393 B CN101504393 B CN 101504393B
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Abstract
The invention discloses an HPLC detection method for a d-biotin and contents of all impurities thereof. At present, the relatively authoritative analytical method is the European Pharmacopoeia, which provides a TLC control method for the d-biotin impurities based on the content detection by a titration method of the American Pharmacopoeia, and the detection method is a semi-quantitative detection method which is low in sensitivity. The chromatographic conditions of the method are that: a reversed-phase C18 column , a mobile phase of trifluoroacetic acid water solution with the mass concentration of 0.05 percent-acetonitrile, the flow rate of between 0.5 and 1.5ml/min, the detection wave length of between 210 and 220nm are adopted; and in the mobile phase, the volume ratio of the trifluoroacetic acid water solution to the acetonitrile is 60-80:40-20; and the impurity containing d-biotin is dissolved and diluted by the mobile phase, a sample is introduced for detection, and the d-biotin and the contents of all the impurities in the d-biotin are detected by a high performance liquid chromatograph. The detection method has the advantages of strong specialization, accurate result and high sensitivity.
Description
Technical field
The present invention relates to the mensuration of content of material, the HPLC assay method of specifically a kind of d-biotin and each impurity content thereof.
Background technology
The d-biotin (d-Biotin) claim biotin again, biotin, and it is a kind of with free or be distributed widely in the animal vegetable tissue with the form of protein bound, the d-biotin has been widely used in the feed addictive aspect of medicine, poultry, domestic animal.Because of the synthetic route of d-biotin is long, in its building-up process, can introduce many impurity (A-H), the research of the analytical approach of relevant d-biotin impurity has also obtained progress faster.Relatively authoritative analytical method is an European Pharmacopoeia at present, it proposes the TLC control method of d-biotin impurity on the basis of the titrimetry assay of American Pharmacopeia, detect impurity in the d-biotin with the TLC method, regulation allows and has only an impurity spot colors size greater than 0.25% own control solution spot, but must not be greater than 0.5% own control spot, the detection of TLC method is limited to 0.25%, and this kind detection method is a half-quantitative detection, and its sensitivity is low.
Summary of the invention
Technical matters to be solved by this invention is to overcome the defective that above-mentioned existing method exists, and provides a kind of HPLC of utilization method to measure the quantivative approach of d-biotin and impurity content thereof.
For this reason, the present invention adopts following technical scheme: the HPLC assay method of d-biotin and impurity content thereof, its chromatographic condition is: adopt anti-phase C
18Post, mass concentration are the moving phase of trifluoroacetic acid aqueous solution-acetonitrile of 0.05%, the flow velocity of 0.5-1.5ml/min, and the detection wavelength of 210-220nm, the volume ratio of trifluoroacetic acid aqueous solution and acetonitrile is 60-80:40-20 in the moving phase; With moving phase dissolving impure d-biotin and dilution, sample introduction is measured, and detects the content of d-biotin and each impurity thereof by high performance liquid chromatograph.Be the structural formula of each impurity below:
This method also is suitable for the assay of all kinds of preparations of d-biotin simultaneously except that being used to measure the content of d-biotin and impurity thereof.
Above-mentioned HPLC assay method is equipped with ultraviolet spectrophotometer on the described high performance liquid chromatograph, the preferred 1.0ml/min of described flow velocity, the preferred 210nm of the wavelength of detection.
Assay method specificity of the present invention is strong, the result is accurate, improved sensitivity greatly, adapt to medicine and register international technical requirement (ICH) about the qualitative and quantitative needs of impurity in the medicine greater than 0.1% content, for the method for setting up HPLC mensuration biotin impurity is laid a good foundation, help to improve the quality control of biotin, important meaning is all arranged guaranteeing its quality and drug safety.
Description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of d-biotin of the present invention and impurity thereof.
Embodiment
1 instrument and reagent
High performance liquid chromatograph (Agilent 1100), day island proper Tianjin UV2201 type ultraviolet spectrophotometer, the N2010 of Zhejiang University series chromatogram data workstation.
D-biotin 3 batch samples (20080901,20080902,20070903) provide by Zhejiang Medicine Co; Water is double distilled water, and impurity A～H all makes by oneself; Acetonitrile is a chromatographically pure, and it is pure that all the other reagent are analysis.
2 methods and result
2.1 chromatographic condition
Chromatographic column: MG-C
18Post (250mm * 4.6mm, 5 μ m); Moving phase: mass concentration is 0.05% trifluoroacetic acid aqueous solution-acetonitrile (75:25); Flow velocity: 1.0ml/min; Detect wavelength: 210nm; Sample size 10 μ l.
2.2 solution preparation
Need testing solution: it is an amount of that precision takes by weighing the d-biotin, with moving phase dissolving and dilution, makes the need testing solution that contains d-biotin 1.0mg/ml.
Contrast liquid: precision is measured need testing solution 1ml, puts in the 100ml measuring bottle, and it is stand-by to add the moving phase constant volume.
Impurity solution: it is an amount of that precision takes by weighing each impurity A～H respectively, dissolves and be diluted to the solution of each component 0.1mg/ml with moving phase.
Mixed solution: precision takes by weighing the d-biotin and each impurity A～H is an amount of respectively, with moving phase dissolving and dilution, makes the mixed solution that contains each component 0.1mg/ml.
2.3 detect the selection of wavelength
Take by weighing the d-biotin respectively and each impurity A～H is an amount of, be mixed with the solution of suitable concn with moving phase, in the interscan of ultraviolet 200～400nm wavelength coverage, the result shows that compound does not all have the feature uv absorption, and therefore, selecting to detect wavelength is 210nm.
2.4 system suitability test
Get the mixed solution sample introduction and measure, the result shows that as figure d-biotin 1 all can separate fully with its each impurity A～H.By the peak of d-biotin, number of theoretical plate reaches 5500, and d-biotin and its each impurity reach baseline separation.
2.5 each impurity location
Get each impurity solution sample introduction, write down each impurity retention time, get need testing solution again and mix respectively with each impurity solution, sample introduction is measured, detect single impurity content and increase, promptly for this reason impurity go out the peak position.
2.6 detectability
Under the chromatographic condition under " 2.1 " item, with moving phase dissolving and progressively dilution, analyze, be 3 o'clock at signal to noise ratio (S/N ratio) S/N, the minimum detection that calculates the d-biotin is limited to 0.5ng (0.01%).
2.7 stability test
Get need testing solution, place respectively 0,6,12,18 and 24h after sample introduction measure, the result shows, d-biotin peak area and have no significant change with each impurity peak area summation number percent, RSD is respectively 0.2% and 0.7%, illustrates that need testing solution is stable in 24h.
2.8 impurity determination
Precision measures " 2.2 " need testing solution and contrast liquid sample introduction is measured, the record chromatogram is to 10 times of d-biotin retention time, the impurity summation that calculates three batch samples with Self-control method is respectively 0.19%, 0.26%, 0.25%, wherein each single impurity all is no more than 0.1%, the results are shown in Table 1.
Table 1 sample dirt content test result (%)
3 conclusions
3.1 in the test gained chromatogram d-biotin and each impurity peaks are carried out UV scanning, all there is not the feature uv absorption, be 210nm so select to measure wavelength, can reach 0.01% through testing its detectability, can adapt to impurity and detect.
Mass concentration is that 0.05% trifluoroacetic acid aqueous solution can effectively strengthen the reservation of d-biotin in post, can effectively improve the peak shape hangover.Under this chromatographic condition, d-biotin peak retention time is suitable, and separablely goes out impurity A, B, C, D, F peak, all goes out the peak before the d-biotin.Behind the adjacent d-biotin of impurity G, the H peak.Unique regrettably impurity E appearance time is located about 10 times of d-biotin peak retention time, taste with the gradient elution test, but because detecting wavelength is 210nm, unstability of base line is fixed, so can't test (see Fig. 1,1 peak among the figure is d-biotin peak) with gradient.
3.2 this method is found the singularity of the biotin of Zhejiang Medicine Co's production because of its technology, B free from foreign meter, C, D, and this method also is suitable for the assay of this product and preparation thereof simultaneously except that being used to check the impurity.
Claims (3)
1.d-the HPLC assay method of biotin and impurity content thereof, its chromatographic condition is: adopt anti-phase C
18Post, mass concentration are the moving phase of trifluoroacetic acid aqueous solution-acetonitrile of 0.05%, the flow velocity of 0.5-1.5ml/min, and the detection wavelength of 210-220nm, the volume ratio of trifluoroacetic acid aqueous solution and acetonitrile is 75: 25 in the moving phase; With moving phase dissolving impure d-biotin and dilution, sample introduction is measured, and detects the content of d-biotin and each impurity thereof by high performance liquid chromatograph;
The structural formula of described impurity is as follows:
2. HPLC assay method according to claim 1 is characterized in that: on the described high performance liquid chromatograph ultraviolet spectrophotometer is housed.
3. HPLC assay method according to claim 1 and 2 is characterized in that described flow velocity selects 1.0ml/min for use, detects wavelength and selects 210nm for use.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102539616B (en) * | 2012-02-14 | 2013-10-23 | 山东师范大学 | Method for extracting and detecting biotin of bird nest |
| CN104614479B (en) * | 2015-01-30 | 2016-06-22 | 北京市营养源研究所 | A kind of detection method of food vitamins |
| CN110887913B (en) * | 2019-12-27 | 2020-10-20 | 上虞新和成生物化工有限公司 | HPLC detection method of biotin intermediate diamino substance |
| CN113125580A (en) * | 2019-12-31 | 2021-07-16 | 深圳奥萨制药有限公司 | High performance liquid chromatography analysis method for determining biotin in complex components |
| CN114371243A (en) * | 2021-12-28 | 2022-04-19 | 湖南中晟全肽生化有限公司 | HPLC detection method of d-biotin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101195629A (en) * | 2006-12-05 | 2008-06-11 | 浙江医药股份有限公司新昌制药厂 | D-biotin purification process |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101195629A (en) * | 2006-12-05 | 2008-06-11 | 浙江医药股份有限公司新昌制药厂 | D-biotin purification process |
Non-Patent Citations (2)
| Title |
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| 宋金春等.HPLC测定注射用13种复合维生素中维生素H、叶酸及维生素B_(12)含量.《中国药学杂志》.2007,第42卷(第17期),1345-1347. * |
| 李兰译.高效液相色谱电化学检测维生素预混料中生物素含量.《国外畜牧学-饲料》.1998,(第04期), * |
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