CN101303334B - HPLC testing method of Nosiheptide product biological valence content - Google Patents
HPLC testing method of Nosiheptide product biological valence content Download PDFInfo
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- 238000012360 testing method Methods 0.000 title claims description 28
- 238000004128 high performance liquid chromatography Methods 0.000 title claims description 26
- FPTCMHOCGKKRGQ-WYOWUDGCSA-N Multhiomycin Natural products CC=C1NC(=O)[C@@H](NC(=O)c2csc(n2)c3cc(O)c(nc3c4csc(n4)[C@H]5CSC(=O)c6[nH]c7cccc(COC(=O)[C@@H](O)C[C@H](NC(=O)c8csc1n8)c9nc(cs9)C(=O)N5)c7c6C)c%10nc(cs%10)C(=O)N[C@@H](C)C(=O)N)[C@H](C)O FPTCMHOCGKKRGQ-WYOWUDGCSA-N 0.000 title claims description 9
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to a high performance liquid chromatographic (HPLC) detecting method of the bioavailability content of nosipeptide preparation, which pertains to the biochemical technical field. Before the nosipeptide preparation is diluted, a mixing solution of an organic solvent and buffer solution is added for extraction; a high performance liquid chromatograph with ultraviolet-visible spectrophotometer is adopted, with a mobile phase with the ratio of acetonitrile: water phase equal to 50: 50, a flow velocity between 0.5ml/min to 2.0ml/min, and an anti-phase C18 macromolecular packing column, and a detecting wavelength between 230nm- 250nm. The areas of various secondary peaks are converted according to the area conversion coefficient (K), and the results are processed by the method for processing the main peaks, and the HPLC determination results are converted by the average conversion of content correction coefficient (P) to obtain the bioavailability content.
Description
Technical field
The invention belongs to technological field of biochemistry, relate to the content method of inspection of that western peptide of a kind of feed medicated premix and various goods thereof, be specifically related to tire high performance liquid chromatography (HPLC) detection method of content of the various product biologicals of that western peptide.
Background technology
The various medicated premixs of interpolation in feed to ward off disease, promote growth, improve efficiency of feed utilization, have become the extensive employing of feed circle and have generally acknowledged one of effective way.Since half a century, people constantly seek better more novel adding ingredient, to overcome deficiency and the defective that traditional composition exists.New that western peptide of veterinary drug (having another name called nosiheptide, English name Nosiheptide) is bionic product, produces by Streptomyces actuosus fermentation, and be a kind of novel non-absorptive-type feeding antibiotic.Newcomer as novel sulphur-containing cyclic polypeptide antibiotics genus, that western peptide has applied widely, broad-spectrum antiseptic, growth promotion is good, addition is few, improve efficiency of feed utilization, alimentary canal does not absorb, the body noresidue and little to environmental impact, take safe, have no drug resistance substantially and do not produce characteristics such as intersect with other microbiotic, especially fabulous adaptability and minimum persistence characteristic are widely used in feed industry, are a kind of more satisfactory feeding antibiotic updated articles.
That western peptide of pure product is a kind of yellow crystal, belongs to liposoluble substance, and its molecular weight is 1222, and molecular formula is C
51H
43N
13O
12S
6, molecular structure (Merck index, fourth edition), be by 12 seed amino acids and derivant form many skins of sulfur-bearing class material, structure is referring to figure below.
Owing to the production of that western peptide is extracted purifying by microbial fermentation product and is obtained, and in fermenting thick thing, it has a large amount of and that western peptide symbiosis, other component that molecular weight and structural formula are all comparatively approximate with it, these components also have certain antibacterial action, and they should belong to the symbiosis component that interrelated relation is arranged with that western peptide.Therefore, when measuring the content of that western peptide, wherein the useful effect of Fei Naxi peptide moiety can not be left in the basket.
Antibiotic kind is a lot, the chemical property difference, and therefore concrete detection method also varies, and can be classified as two classes but after all get up: chemical assay and microbial detection method (immunoassay).
Microbial detection method (immunoassay) is the general special efficacy method of inspection that microbiotic is generally acknowledged both at home and abroad, and its result can reflect antibacterial material compounding ingredients in the antibiotic or claim the comprehensive effect of multiple component.That western peptide biological value content standing order also is appointed as the microbial detection method and is come assessment result.
But owing to many reasons, cause the poor practicability of present regulation method of testing, waste time and energy and measurement result precision difficulty reaches requirement.At first, its concrete checkout procedure is longer experimental period, and the experiment of single sample needs time spent average out to 18 hours; Secondly, the utilization factor average out to 60%-70% of the experiment material of single experiment, experiment material consumption waste is bigger; Moreover the method fiducial limit rate of measurement result 〉=5% probability average out to occurs more than 80%.
Summary of the invention
The objective of the invention is the problems referred to above, adopt the HPLC assay method of quicklook,, that western peptides products content results can directly be expressed as biological value content by special processing at the prior art existence.
The present invention includes following content:
The high performance liquid chromatography of Nosiheptide product biological valence content (HPLC) detection method comprises the pre-service of testing sample, and the chromatographic condition of appointment is measured the Treatment Analysis of collection of illustrative plates etc.:
(1) uses and have the mixed solution of quite fat-soluble organic solvent and damping fluid that its preparation is extracted,, measure required liquid to be measured thereby obtain HPLC so that can extract that western peptide fully to that western peptide;
(2) chromatographic condition of appointment is: the high performance liquid chromatograph of band UV, visible light spectrophotometric detector, second cyanogen: the moving phase of water=50: 50; 0.5ml/min the flow velocity of～2.0ml/mim; Anti-phase carbon 18 big molecule packed columns; The detection wavelength of 230nm-250nm;
(3) will measure collection of illustrative plates each submaximum area with area conversion factor (K) to its conversion after, with the method result of handling main peak, this area conversion factor (K) is: 1/2～3/4 again;
(4) with the mean value conversion acquisition biological value content of HPLC measurement result by content compensation coefficient (P), content compensation coefficient (P) is 0.970～1.050.
Organic solvent in the mixed solution of extraction usefulness of the present invention is meant N, dinethylformamide (N, N-dimethylformamide DMF), and damping fluid is meant phosphoric acid-phosphate buffer.
More than the described water of (2) be the phosphate aqueous solution of≤0.50% percentage by weight.
In sum:
1. the present invention has narrated that the western peptide of mixed solution high efficiency extraction that adopts organic solvent and damping fluid, to obtain to treat the preprocess method of test sample extract.This treating fluid can be used for that western peptides products assay or is used for other non-mensuration purpose.
2. measure the method for that western peptides products major component and relevant other component content under the HPLC condition that has been described in detail in appointment of the present invention, this method is practical fast, disturb less, the measurement result good reproducibility, is an effective reliable novel assay method.
3. the present invention can directly represent the method for Nosiheptide product biological valence content with the HPLC measurement result after processing, and adopts computing method effectively and reliably when measurement result is handled.
Embodiment
The detecting instrument that the present invention relates to, condition, method step are as follows:
1., inspection machine condition:
High performance liquid chromatograph (band UV, visible light spectrophotometric detector)
Moving phase: second cyanogen: water (phosphate aqueous solution of≤0.50% percentage by weight)=50: 50;
Flow velocity: 0.5ml/min-2.0ml/mim.
Test column: anti-phase carbon 18 big molecule packed columns.
Detect wavelength: 230nm-250nm.
2., test liquid pre-service:
The standard stock solution:
The accurate standard items that claim fixed known that western peptides products content (or known suitable biological value) add that the DMF liquid of 20ml is ultrasonic to be diluted to the volumetric flask full scale to thoroughly dissolving with DMF liquid in suitable volumetric flask, it is prepared into the stock solution of 1000ppm.Lucifuge cryopreservation, its term of validity are three months.
The standard test liquid:
After above-mentioned standard stock solution returns to room temperature, accurately pipette and calculate required volume number in advance, and place suitable volumetric flask, the DMF that adds 10ml, being mixed to spare then is diluted to the volumetric flask full scale with methanol solution, it is prepared into the test liquid of 50ppm, or is mixed with dilution series liquid.
With microfiltration membrane filtration mistake, measure usefulness in order to the HPLC check.This liquid should face time spent preparation, and lucifuge as far as possible.
Treat the sample test liquid:
The accurate title, decided the suitable quality that calculating in advance prepares the needed sample to be checked of 50ppm concentration, places the volumetric flask of appropriate volume, adds the DMF liquid liquid of 40ml, and the monophosphate monophosphate salt buffer of 10ml, and sample is fully wetting.With ultrasonic or extract, be diluted to the volumetric flask full scale with DMF liquid liquid finishing after under the room temperature, treat the sample test liquid into about 50ppm and it is prepared with the jolting method.With microfiltration membrane filtration mistake, measure usefulness for the HPLC check.This liquid should face time spent preparation, and lucifuge as far as possible.For guaranteeing degree of confidence, same lot number sample should carry out three or the above parallel sample of equal processing.
3., sample HPLC measures:
Open the HPLC instrument, according to the present invention 1. the test condition of appointment set, the prerun instrument is to obtain baseline stably.To the standard test liquid with treat sample test liquid operation sample introduction, the each sample introduction 20ul of each sample liquid is to obtain corresponding chromatogram spectrogram with the micro-injection injector.
4., the computing method of measurement result:
Handle calculating with peak area external standard working curve method or with single-point peak area external standard method.
5., different time goes out the treatment principle of peak component in measurement result is calculated:
When handling that western peptide peak of main peak, can be directly with the peak area external standard method of that Xi Taifeng of standard (working curve method or with the Single-point Calculating Method) calculating of comparing, in the hope for the treatment of the content of that western peptide in the sample.
When handling other component (other component peaks of relevant property with main peak, submaximum) of Fei Naxi peptide, after the area of each submaximum can being converted with area conversion factor (K) earlier, handle with handling the equal computing method of that Xi Taifeng again.
Directly calculating the content results that obtains other submaximum with the standard items main peaks, verified and calculate respectively between separately the result with the pure product of these components and to have certain difference, but this discrepancy is very little, and almost can ignore.
Generally speaking, two to three submaximums can occur, but have only one near the submaximum of main peak the calculating meaning to be arranged just.Promptly the result of this submaximum can produce certain influence to net result, and other submaximum result all can ignore.
Can occur the situation of a plurality of big submaximums in special sample, this moment, all submaximums should be recomputated consideration to the influence of final measurement result.
6., after obtaining the HPLC result of that western peptide, can directly adopt content compensation coefficient (P) to calculate its biological value content expression values.
Feasibility analysis to the present invention program is as follows:
1, treat the representative studies of sample test liquid to composition to be determined in the sample:
Handling with the mixed liquor high efficiency extraction of using among the present invention treats that the method for test sample obtains by following research.
Research theory shows: no matter adopt what method to carry out assay, the pre-treatment of sample is crucial, requirement be pre-treatment obtain treat the sample test liquid real content value of the composition to be measured of representative sample stably.It is the recovery big (〉=95%) for the treatment of fluid and can keep certain stability (RSD, CV≤2%) time, this method just can be a reliable quantitative test method.
Because the macromolecular structure of that western peptide has determined it that extraction conditions is had suitable susceptibility, influencing the factor that western peptide is extracted efficiently and stably has a lot, as temperature, time, pH, type of solvent etc., all can exert an influence to its dissolution rate or the stability after stripping.Pre-treatment process or condition are controlled badly, very easily cause and extract not exclusively or form degraded.We extract compare operation with the pre-mixing agent of different actual contents by different condition, and carry out statistical study.Efficient statistical research by influence factor shows: the difference of pre-treatment condition can cause diverse extract concentration situation, thereby causes final testing result difference.Have only and used suitable treatment conditions, could obtain higher alluvial.
Experimental results show that:
1., adopt the mixed solution of organic solvent and buffering solution to handle, just can reach efficiently and extract.
2., different organic solvent effect difference that fat-soluble that western peptide is extracted.
3., adopting appropriate organic solvent (DMF) to mix with the damping fluid that suits extracts that western peptide, just can obtain the highest extraction value and the most stable extraction efficiency.
Data research shows: the average recovery rate value for the treatment of the sample test liquid that this extracting method obtains reaches 102.6%, satisfies the method requirement of 90%-110%; The CV of this average recovery rate (%) is 0.6%, is significantly less than 2% theoretical method regulation.
2, method of testing reliability consideration of the present invention:
The method condition combination of using among the present invention obtains by following research.
The method research theory shows: the feasibility of detection method and reliability are the keys of decision testing result.Therefore need carry out the evaluation of science to it, liquid chromatogram measuring method be verified below by method precision, accuracy, the range of linearity:
1., precision:
Method precision is meant the repeatability or the reappearance of analysis result.Quantivative approach for a practicality, its repeated precision (both coefficient of variation CV%, RSD%) must be controlled in the as far as possible little scope, consider that the outer precision size of method itself and operation factors is also relevant with testing concentration, and general index is if concentration when 〉=100mg/kg, CV (%) should≤5%.
We are that certain lot number formulation samples of 0.5% is carried out the collimation experiment with theoretical content, detect 40 parallel samples altogether.The testing research digital proof shows the CV (%)=0.539% that detects the sample test result who obtains under condition of the present invention, is significantly less than 5% requirement, illustrates that the repeated test precision of this method reaches the theoretical method regulation fully.
2., accuracy:
The accuracy of method is meant the degree of closeness of measured value and actual value, adopts the interpolation recovery to represent usually.As an available quantivative approach, the recovery generally should be in 80%～110% scope.And as a quantivative approach efficiently, the recovery should be in being controlled at 95%～105% scope, and the precision of its recovery should remain on below 2%.
The test data that reaches different actual concentration by the repeatedly repeated test to same concentration sample studies show that, this method is in that western peptide concentration from 0.1% to 96.0% scope, precision is the poorest as a result is 0.30% in its sample replicate determination, satisfies≤2% requirement; The preparation recovery of variable concentrations remains in the scope of 102%-104%, and the recovery precision of each concentration the poorest be 1.24%, satisfy theoretical method regulation.
3., the range of linearity:
The range of linearity of method has been represented the popularity that this method can the application of samples test, and the sample in this range of linearity uses this method to test, and linear the guarantee arranged.Effective range of linearity of an effective quantitative analysis method should guarantee statistics linearly dependent coefficient r 〉=0.995 of 5 concentration at least, and should guarantee precision RSD≤5% of the variable concentrations recovery.
Studies show that by test data that western peptide sample of nearly 15 different actual concentration in the scope of that western peptide concentration from 0.1% to 96.0%, the recovery and recovery CV keep meeting outside the requirement of method regulation, its linearly dependent coefficient r=0.999984% satisfies the theoretical method regulation.Therefore think that the mensuration of the Nosiheptide product biological valence content of this method in this scope all is suitable for.
3, the processing at other related component peak (submaximum) in the mensuration:
The treatment principle of measurement result obtains by following research among the present invention.
Owing to again through the acquisition of purifying someway, so exist other component in that western peptide after that western peptide is cultivated by microbial fermentation.
When the biological value content of correct that western peptide of expression, should consider that western peptide is a kind of biological antibiotic element, the effect of the related component of its symbiosis or coexistence should be not out in the cold, should take into full account the resultant effect of that western peptide and other each component common performance in antibiotic.And the result who obtains when measuring the biological value content of that western peptide with immunoassay (microbial detection method) must be the acting in conjunction result of that western peptide and its symbiosis component.These components are different at the western peptide of character, molecular structure or molecular weight and that, but they and that western peptide exist interdependence, or similar or very approaching.And these components also show certain antibacterial action, but will hang down compared with the functioning efficiency of that western peptide.
When measuring that Xi Taifeng (main peak) with the HPLC method, these components also can go out the peak, generally are referred to as submaximum at this.
In theory, should come its content results of these components of difference quantitative Analysis respectively with the pure product of these components, but the acquisition of the pure product of these components is very difficult, or even almost impossible.Therefore have that western peptide main peak of employing come direct quantitative they, be a kind of easier method, key is can cause great error at measurment thus.By studies show that, the result after adopting this short-cut method to handle, very little with the discrepancy of actual value, even almost can ignore.
Therefore, we can adopt following principle to calculate when handling these submaximums:
1., can be directly directly come the quantitative Analysis submaximum and obtain its result with the main peak of western poly saccharide peptide standard product.After the area of each submaximum can being converted its area with certain area conversion factor (K) earlier, the equal disposal route method of handling with main peak is handled again.And the concrete numerical value of this area conversion factor (K) is obtained by the HPLC result and the microbial detection method result of more same concentration sample.By research, we obtain this area conversion factor (K) and generally exist: in the scope of 1/2-3/4.
2., two to three submaximums can occur generally speaking, but have only one near the submaximum of main peak the calculating meaning to be arranged relatively just, other peak is minimum, and promptly the result of this submaximum can produce certain influence to net result, and other submaximum result all can ignore.
3., under special circumstances, the situation of a plurality of big submaximums can occur, should rethink how to convert the influence of all submaximums to final measurement result this moment.
4, the conversion of that western peptide HPLC content and biological value content:
The biological value content of that western peptide comes the method for converting expressing to obtain by following research among the present invention with HPLC content.
Because biological value can reflect the resultant effect of antibiotic more exactly, antibiotic measurement result generally requires to express with biological value content, and the net result of that western peptide of sulfur-bearing polypeptide antibiotics can record its biological value content with biologic assay.
The HPLC measurement result of that western peptide has been expressed the quality percentage of that western peptide and other composition in the preparation.Because the nosiheptide preparation antibacterial effect is the ability of being expressed by that western peptide in the preparation and other component, when that western peptide percent increased in the preparation, its antibacterial effect can strengthen, and simultaneously, other component percent increases also can partly strengthen its antibacterial effect.
If there is fixing relevance between this heavy percentage composition and its biological value content, promptly both ratio values of being maintained fixed then can directly be converted into its biological value content by the result of HPLC.Whether this association can remain a relatively-stationary coefficient by the ratio of investigating both, and this coefficient has high precision and proved.
Show that by outcome research this ratio P (content compensation coefficient) exists really to two kinds of method of testings of variable concentrations sample.When changing in the scope of formulation concentrations at 0.1%-96.0%, result of study shows that content compensation coefficient (P) fluctuation is very little, and the coefficient of dispersion CV of content compensation coefficient (P) is 0.954%, be significantly less than 5% theoretical method requirement, this illustrates that western peptide biological value content can directly obtain with the mean value conversion of HPLC measurement result by content compensation coefficient (P).General content compensation coefficient (P) remains in the scope of 0.970-1.050.
The present invention adopts the HPLC assay method of quicklook, by special processing, that western peptides products content results can directly be expressed as biological value content, this has just shortened the time widely, alleviated labour intensity, reduced the consumption of experiment material, therefore bigger practicality and economic worth have been arranged.
Claims (1)
1. the high performance liquid chromatography of Nosiheptide product biological valence content (HPLC) detection method comprises the pre-service of testing sample, the chromatographic condition of appointment, and the Treatment Analysis of mensuration collection of illustrative plates is characterized in that:
(1) uses and have the mixed solution of quite fat-soluble organic solvent and damping fluid that its preparation is extracted, obtain that western peptide HPLC liquid to be measured that western peptide; Described have quite fat-soluble organic solvent to be meant N to that western peptide, dinethylformamide, and the damping fluid of mixing is meant phosphoric acid-phosphate buffer;
(2) chromatographic condition of appointment is: the high performance liquid chromatograph of band UV, visible light spectrophotometric detector, acetonitrile: the moving phase of water=50: 50; 0.5ml/min the flow velocity of～2.0ml/mim; Anti-phase carbon 18 big molecule packed columns; The detection wavelength of 230nm-250nm; Described water is the phosphate aqueous solution of≤0.50% percentage by weight;
(3) area that will measure each submaximum of collection of illustrative plates with area conversion factor (K) to its conversion after, with the method result of handling main peak, this area conversion factor (K) is: 1/2～3/4 again;
(4) with the mean value conversion acquisition biological value content of HPLC measurement result by content compensation coefficient (P), content compensation coefficient (P) is 0.970～1.050;
(5) above-mentioned testing sample is fermenation raw liquid or its various preparations of that western peptide.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008100624175A CN101303334B (en) | 2008-06-06 | 2008-06-06 | HPLC testing method of Nosiheptide product biological valence content |
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|---|---|---|---|
| CN2008100624175A CN101303334B (en) | 2008-06-06 | 2008-06-06 | HPLC testing method of Nosiheptide product biological valence content |
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| CN101303334A CN101303334A (en) | 2008-11-12 |
| CN101303334B true CN101303334B (en) | 2011-08-24 |
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| CN1840684A (en) * | 2006-01-26 | 2006-10-04 | 浙江工商大学 | Process for fermentation preparation of nosiheptide by using streptomycete |
| CN1899599A (en) * | 2005-07-19 | 2007-01-24 | 复旦大学 | Method for preparing Nosiheptide premixed agent |
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| CN1899599A (en) * | 2005-07-19 | 2007-01-24 | 复旦大学 | Method for preparing Nosiheptide premixed agent |
| CN1840684A (en) * | 2006-01-26 | 2006-10-04 | 浙江工商大学 | Process for fermentation preparation of nosiheptide by using streptomycete |
Non-Patent Citations (11)
| Title |
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| 何建勇.兽用抗生素诺西肽(nosiheptide)的研究进展.《药物生物技术》.2004,第11卷(第6期),406-409. |
| 冯美卿 |
| 周珮.那西肽脂质体的制备及其体外抗乙肝病毒的研究.《药学学报》.2005,第40卷(第5期),462-465. |
| 楼双凤 |
| 王晶 |
| 蔡钦生 |
| 蔡钦生;黄海;冯美卿;周珮.那西肽脂质体的制备及其体外抗乙肝病毒的研究.《药学学报》.2005,第40卷(第5期),462-465. * |
| 韩果红 |
| 韩果红.诺西肽发酵工艺的研究.《中国优秀硕士学位论文全文数据库》.2007,(第1期),1-103. * |
| 韩果红;楼双凤;王晶;何建勇.兽用抗生素诺西肽(nosiheptide)的研究进展.《药物生物技术》.2004,第11卷(第6期),406-409. * |
| 黄海 |
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