CN103776913A - HPLC method for quick teicoplanin fermentation liquor unit detection - Google Patents
HPLC method for quick teicoplanin fermentation liquor unit detection Download PDFInfo
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- CN103776913A CN103776913A CN201210442647.0A CN201210442647A CN103776913A CN 103776913 A CN103776913 A CN 103776913A CN 201210442647 A CN201210442647 A CN 201210442647A CN 103776913 A CN103776913 A CN 103776913A
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Abstract
The invention discloses an HPLC method for quick teicoplanin fermentation liquor unit detection. The method comprises the following steps: processing teicoplanin fermentation liquor with alkali, performing ultrasonography, performing centrifugation for two times, enabling clear liquor to pass a filtration film at one step, and then placing the filtrated liquor on a machine to be detected and analyzed, wherein a high-performance liquid chromatograph (HPLP) is mainly equipped with a reversed-phase C18 column; a detector is an ultraviolet detector; a mobile phase is an ammonium acetate solution and an acetonitrile buffer solution for gradient elution, and data is collected and analyzed at a condition of 277 nm. The detection method provided by the invention is simple, convenient and fast, and is high in sensitivity and good in accuracy.
Description
Technical field
The invention belongs to detection method technical field, in particular to the HPLC method for quick of a kind of teicoplanin fermentation liquor unit.
Background technology
Teicoplanin belongs to glycopeptide antibiotics, gram-positive bacteria is had to powerful antibacterial activity, be usually used in clinically serious gram positive bacteria infection, the effect of golden Portugal bacterium is often better than to glycopeptide antibiotics vancomycin, therefore the production of teicoplanin comes into one's own day by day with research and development.Teicoplanin is that external Parenti etc. obtains from a strain actinoplanes A.teichomyceticus nov.sp. fermentation the earliest, the i.e. conventional ATCC31121 now of this bacterial classification; Domestic research mainly contains rare actinomycete SIIA92-363, A.teichomyceticus 98-1-227 and SIIA 05-03-136 thereof, all separable to teicoplanin in the fermentation liquor of this three strains bacterium.Research finds that this medicine is a complex set of compound, by 5 TA that structure is very close
2-1, TA
2-2, TA
2-3, TA
2-4and TA
2-5composition.The 6th active substance is TA
3-1, this compound is not present in fermentation liquor, but in extraction and purifying, all has trace to exist.
Along with the raising day by day of fermentation technique, the detection analysis of teicoplanin fermentation liquor unit also becomes increasingly conspicuous in producing and researching and developing.At present conventional fermentation liquor unit to detect be high performance liquid chromatography (HPLC), with respect to biological detection or additive method, HPLC method has express-analysis, the advantage such as quantitatively accurate, so application is also increasingly extensive.Conventional HPLC method is the detection method of calendar year 2001 the 14 edition Japanese Pharmacopoeia (JP XIV) regulation at present, be specially: mobile phase A: 15% acetonitrile-sodium dihydrogen phosphate (0.025mol/L), B:70% acetonitrile-sodium dihydrogen phosphate (0.025mol/L), two-phase all regulates pH to 6.0 with 1mol/L NaOH solution, gradient elution, flow velocity is 1.8mL/min.The method shortcoming is: each sample analysis time is 60min, is not easy to fast detecting; Flow velocity is higher, and long-time use can cause the voltage rise of chromatographic column post high, increases pump loss, damages chromatographic column, the accuracy of impact analysis and sensitivity.
Summary of the invention
In view of the deficiencies in the prior art, the object of the invention is to shorten the analysis time of HPLC method detection teicoplanin fermentation liquor unit, improve separating effect and the serviceable life of performance liquid chromatographic column simultaneously, thereby the method for a kind of fast detecting teicoplanin fermentation liquor unit is provided.
In order to realize object of the present invention, inventor studies by lot of experiments, finally obtains following technical scheme:
A HPLC method for quick for teicoplanin fermentation liquor unit, comprises the steps: to get teicoplanin fermentation liquor, by extremely alkalescence of NaOH solution adjusting pH, after ultrasonic 0.2-1h centrifugal 1-3 time, get supernatant, excessively after 0.22 μ m filter membrane, carry out HPLC detection, testing conditions is:
(1) chromatographic column: the anti-phase C18 post take octadecylsilane chemically bonded silica as filling agent;
(2) detecting device: UV-detector;
(3) detect wavelength: λ=277nm;
(4) mobile phase: A:0.01mol/L ammonium acetate solution, B:90% acetonitrile solution, filters ultrasonic rear use, gradient elution; The concentration of polar organic solvent is that continuity increases progressively, and 0min to 17min is incremented to 50% by 5%, and 17min to 25min is decremented to 5% by 50%;
(5) flow velocity: 1.0mL/min;
(6) column temperature: 25 ℃;
(7) sampling volume: 20 μ L.
Preferably, the HPLC method for quick of described teicoplanin fermentation liquor unit, comprise the steps: to get teicoplanin fermentation liquor, with NaOH solution adjusting pH to 8-12, ultrasonic 30min is placed in centrifuge tube, centrifugal 10min under 10000rpm, get supernatant and be placed in another centrifuge tube, under 10000rpm, centrifugal 10min, gets supernatant, cross 0.22 μ m filter membrane, carry out HPLC detection by described testing conditions.
Further preferably, above-mentioned teicoplanin fermentation liquor is carried out in pretreatment process, adopting concentration is the pH to 10 that 10%NaOH solution regulates teicoplanin fermentation liquor.
After utilizing method of the present invention to detect teicoplanin fermentation liquor, teicoplanin density calculating method is: be made into the titer of certain gradient concentration with teicoplanin standard items, 20 μ L volume sample introductions, with A
25 component peaks area sum-concentration are done linear regression, draw equation of linear regression, utilize the content of teicoplanin in the calculated by peak area fermentation liquor of teicoplanin in this equation and sample.
Compared with prior art, detection method of the present invention is simple and efficient, highly sensitive, and accuracy is good.Through test, the range of linearity is within the scope of 0.25～5.0mg/mL, and related coefficient is all more than 0.999.Sample detection selectivity is strong, and every methodology index all can meet the detection needs of teicoplanin in fermentation liquor.
Accompanying drawing explanation
Fig. 1 teicoplanin standard items chromatogram;
The fermentation broth sample chromatogram of Fig. 2 embodiment 1.
Embodiment
Below in conjunction with specific experiment, the present invention is further elaborated, but do not limit the present invention.
1, sample treatment
Getting a certain amount of fermentation liquor, is that 10%NaOH solution regulates pH to 10 by concentration, and ultrasonic 30min is placed in centrifuge tube, centrifugal 10min under 10000rpm, gets supernatant and is placed in another 10mL centrifuge tube, centrifugal 10min under 10000rpm, get supernatant, excessively upper machine testing after 0.22 μ m filter membrane.
2, chromatographic condition
(1) chromatographic column: the anti-phase C18 post take octadecylsilane chemically bonded silica as filling agent;
(2) detecting device: UV-detector;
(3) detect wavelength: λ=277nm;
(4) mobile phase: A:0.01mol/L ammonium acetate solution, B:90% acetonitrile solution, filters ultrasonic rear use, gradient elution; The concentration of polar organic solvent is that continuity increases progressively, and 0min to 17min is incremented to 50% by 5%, and 17min to 25min is decremented to 5% by 50%;
(5) flow velocity: 1.0mL/min;
(6) column temperature: 25 ℃;
(7) sampling volume: 20 μ L
3, standard items storing solution
Precision takes 100mg teicoplanin standard items and is placed in 10mi volumetric flask, is settled to scale with 50% ethanol, shakes up, and is mixed with the standard reserving solution that concentration is 10mg/ml, is placed in 4 ℃ of refrigerators for subsequent use.
4, typical curve
With 50% ethanol by the standard items storing solution under 3 be diluted to 0.25,0.5,1.0,2.0,5.0mg/mL, get 20 μ L volume sample introductions under each concentration, with A
25 component peaks area sum-concentration are done linear regression, draw equation of linear regression, utilize the content of teicoplanin in the calculated by peak area fermentation liquor of teicoplanin in this equation and sample.
5, precision
By the teicoplanin standard solution continuous sample introduction of 1.0mg/mL 5 times, each sample introduction 20 μ L, measure according to the chromatographic condition under 2, record peak area, calculate precision.
6, replica test
Get same batch fermentation liquid, obtain 5 samples according to the sample treatment processing in 1, sample introduction is also measured according to the chromatographic condition under 2, calculates relative standard deviation, relatively reappearance.
7, average recovery test
In the fermentation liquor of known content, precision adds teicoplanin standard items respectively, makes that its concentration is 1.0,2.0, each 3 parts of 4.0mg/mL, according to the sample treatment processing under 1, measures calculate recovery rate according to the chromatographic condition under 2.
8, stability test
Get under the teicoplanin standard solution room temperature of 1.0mg/ml and place, the sample introduction respectively at 0,2,4,8,12, under 24h, each sample introduction 20 μ L, measure according to the chromatographic condition under 2, check the stability of sample solution in 24h.
9, sample determination
Get the fermentation liquor of cultivating 100h after culture transferring in conical flask, process according to the sample treatment under 1, measure according to the chromatographic condition under 2, calculate medicine peak area, bring in corresponding typical curve equation, draw as calculated the concentration of teicoplanin.
10, interpretation of result
According to the method preparation standard curve under 4, with A
25 component peaks area sum-concentration are done linear regression, draw equation of linear regression Y=2596000X+1200 (Y is peak area, and X is drug concentration mg/mL), and linearly dependent coefficient is R
2=0.9999, visible teicoplanin is good in 0.25～5.0mg/mL concentration range internal linear.
After 5 sample introductions of precision test, be 1.1% according to calculated by peak area relative standard deviation, show that the method precision is better; After 5 sample introductions of replica test, be 1.5% according to calculated by peak area relative standard deviation, show the method favorable reproducibility; After application of sample, measure the recovery, obtaining the teicoplanin recovery through calculated by peak area is 99.3%, shows that the method authenticity is higher; In stability test, after tested, be 0.9% according to calculated by peak area relative standard deviation, show that this sample standard liquid is placed 24h at ambient temperature more stable.
Get upper machine testing after the fermentation liquor treatment of cultivating 100h after culture transferring, calculate peak area offspring and enter equation of linear regression, recording concentration is 1.3mg/mL.
Can be found out by above test findings, the method accuracy high precision is good, and the indexs such as the recovery, linear relationship, stability test all can meet the fast detecting of teicoplanin in normal fermentation liquid.
Claims (3)
1. a HPLC method for quick for teicoplanin fermentation liquor unit, is characterized in that: get teicoplanin fermentation liquor, by extremely alkalescence of NaOH solution adjusting pH, after ultrasonic 0.2-1h centrifugal 1-3 time, get supernatant, excessively after 0.22 μ m filter membrane, carry out HPLC detection, testing conditions is:
(1) chromatographic column: the anti-phase C18 post take octadecylsilane chemically bonded silica as filling agent;
(2) detecting device: UV-detector;
(3) detect wavelength: λ=277nm;
(4) mobile phase: A:0.01mol/L ammonium acetate solution, B:90% acetonitrile solution, filters ultrasonic rear use, gradient elution; The concentration of polar organic solvent is that continuity increases progressively, and 0min to 17min is incremented to 50% by 5%, and 17min to 25min is decremented to 5% by 50%;
(5) flow velocity: 1.0mL/min;
(6) column temperature: 25 ℃;
(7) sampling volume: 20 μ L.
2. the HPLC method for quick of teicoplanin according to claim 1 fermentation liquor unit, it is characterized in that: get teicoplanin fermentation liquor, with NaOH solution adjusting pH to 8-12, ultrasonic 30min is placed in centrifuge tube, and centrifugal 10min under 10000rpm gets supernatant and is placed in another centrifuge tube, centrifugal 10min under 10000rpm, get supernatant, cross 0.22 μ m filter membrane, carry out HPLC detection by described testing conditions.
3. the HPLC method for quick that will go to the teicoplanin fermentation liquor unit described in 2 according to right, is characterized in that: be the pH to 10 of 10%NaOH solution adjusting teicoplanin fermentation liquor by concentration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104910259A (en) * | 2015-06-02 | 2015-09-16 | 苏州纳微科技有限公司 | Chromatographic purification method for teicoplanin |
| CN111879877A (en) * | 2020-08-10 | 2020-11-03 | 北京卫新医药技术发展中心 | Application of methylparaben in teicoplanin quantitative analysis and use method |
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| CN1741810A (en) * | 2002-08-30 | 2006-03-01 | 鲁汶天主教大学研究开发部 | Glycopeptide antibiotic derivatives |
| CN101423547A (en) * | 2007-10-31 | 2009-05-06 | 浙江医药股份有限公司新昌制药厂 | Purification method of teicoplanin |
| CN102718843A (en) * | 2012-06-30 | 2012-10-10 | 华北制药集团新药研究开发有限责任公司 | Preparation method of single teicoplanin components |
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Patent Citations (3)
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| CN1741810A (en) * | 2002-08-30 | 2006-03-01 | 鲁汶天主教大学研究开发部 | Glycopeptide antibiotic derivatives |
| CN101423547A (en) * | 2007-10-31 | 2009-05-06 | 浙江医药股份有限公司新昌制药厂 | Purification method of teicoplanin |
| CN102718843A (en) * | 2012-06-30 | 2012-10-10 | 华北制药集团新药研究开发有限责任公司 | Preparation method of single teicoplanin components |
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| 付靖, 刘崧, 暴娜, 郑煜: "高效液相色谱法检测替考拉宁组分及其有关物质", 《河北化工》, vol. 29, no. 8, 31 August 2006 (2006-08-31), pages 62 - 63 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104910259A (en) * | 2015-06-02 | 2015-09-16 | 苏州纳微科技有限公司 | Chromatographic purification method for teicoplanin |
| CN111879877A (en) * | 2020-08-10 | 2020-11-03 | 北京卫新医药技术发展中心 | Application of methylparaben in teicoplanin quantitative analysis and use method |
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