CN105738532B - A kind of detection method for zymotic fluid potency of Ledermycining - Google Patents

A kind of detection method for zymotic fluid potency of Ledermycining Download PDF

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CN105738532B
CN105738532B CN201610332799.3A CN201610332799A CN105738532B CN 105738532 B CN105738532 B CN 105738532B CN 201610332799 A CN201610332799 A CN 201610332799A CN 105738532 B CN105738532 B CN 105738532B
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potency
zymotic fluid
ledermycining
sample
detection method
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CN105738532A (en
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陈红丽
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Ningxia Tairui Pharmaceutical Co Ltd
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Ningxia Tairui Pharmaceutical Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

The present invention relates to a kind of detection method for zymotic fluid potency of Ledermycining, feature is to detect the zymotic fluid test sample that Ledermycins using high performance liquid chromatography, then calculates its potency using external standard method.The present invention is detected using high performance liquid chromatography to zymotic fluid potency of Ledermycining, its method is easy, quick, it is easily operated, as a result it is accurate, it is reproducible, high sensitivity, when methyl Aureomycin fermentation liquor potency is between 10 200 u/ml, there is preferable linear relationship, its linearly dependent coefficient is 0.9991.

Description

A kind of detection method for zymotic fluid potency of Ledermycining
Technical field
The invention belongs to chemical analysis technology field, more particularly to a kind of detection side for zymotic fluid potency of Ledermycining Method.
Background technology
Ledermycin, alias demeclocycline, demethyl duomycin, DMCT.This product antimicrobial spectrum is similar to aureomycin, The effect of being characterized in is stronger than tetracycline, terramycin, more stable than aureomycin.It is clinically used for pneumonia, urinary tract infections, gonorrhoea, bacillary Dysentery, brucellosis and children's scarlet fever etc..
The antibacterial activity that Ledermycins is strong compared with tetracycline, absorbs preferable.It is broad-spectrum antibiotic, plays bacteriostasis.It is anti- Bacterium spectrum includes many Grain-positives and negative bacterium, Richettsia, mycoplasma, Chlamydia, actinomyces etc..Now it is mainly used in rickettsia Body disease, brucellosis, poradenia, Eaton agent pneumonia, spirochetosis, chlamydiosis, it can also be used to the blue sun of sensitive leather Property coccus or gram negative bacilli caused by mild infection.
The structure Ledermycined is as follows：
The detection method of the current zymotic fluid that Ledermycins uses AAS to be detected, and adds certain It is measured after ferric trichloride developer, trouble is handled in detection process, and developing time is grown, ferric trichloride nature It is unstable.Therefore develop it is a kind of can quickly, Accurate Determining Ledermycin zymotic fluid potency method always need to be solved Certainly the problem of.
The content of the invention
The defects of it is an object of the invention to overcome above-mentioned prior art, there is provided a kind of method is easy, quick, is easy to grasp Make, as a result accurately, the detection method of reproducible zymotic fluid potency of Ledermycining.
A kind of detection method for zymotic fluid potency of Ledermycining, it is characterised in that using high performance liquid chromatography to going Methyl Aureomycin fermentation liquor test sample is detected, and then calculates its potency using external standard method.
The chromatograph condition of the high performance liquid chromatography is：
Chromatographic column：C18 posts；
Detector：DAD detectors；
Mobile phase：Acetonitrile：0.5 % phosphoric acid solutions=20-30：80-70；
Flow velocity：0.80-1.20 ml/min；
Sample size：10-20 ul；
Detection wavelength：250-260 nm.
The specification of the C18 posts is：The mm of length 250 or 150 mm, the mm of internal diameter 4.6, the um of packing material size 5.
The zymotic fluid that Ledermycins is configured to test sample again after first being dissolved with 0.15 mol/L hydrochloric acid solutions.
It is described dissolved with hydrochloric acid solution after methyl Aureomycin fermentation liquor potency in 10-200 u/ml.
The present invention is detected using high performance liquid chromatography to zymotic fluid potency of Ledermycining, its method simplicity, Quickly, it is easily operated, as a result accurately, reproducible, high sensitivity, when methyl Aureomycin fermentation liquor potency is in 10-200 u/ml Between when, have preferable linear relationship, its linearly dependent coefficient be 0.9991.
Brief description of the drawings
Fig. 1 be Ledermycin zymotic fluid potency reference substance HPLC detection collection of illustrative plates；
Fig. 2 be Ledermycin zymotic fluid potency HPLC detection collection of illustrative plates；
Fig. 3 is the Ledermycin linear relationship chart and regression equation of zymotic fluid potency.
Embodiment
In order that those skilled in the art is better understood from the technical scheme of invention, with reference to specific implementation method pair Technical scheme of the present invention is described in further detail.
Embodiment 1
The zymotic fluid that Ledermycins analyzes the preparation of sample：
The preparation of standard solution：Zymotic fluid potency reference substance 10 mg of Ledermycining is weighed, it is accurately weighed, put 100 In ml volumetric flasks, add flowing phased soln and be settled to scale, shake up.Organic filter mistake of the solution through 0.45um is taken with syringe Filter, obtains reference substance solution.
The preparation of need testing solution：Zymotic fluid 1.0 g of Ledermycining is weighed, it is accurately weighed, it is placed in 100 ml capacity In bottle, add 0.15 mol/L hydrochloric acid solution appropriate, scale is settled to after dissolving, is shaken up, after placing 10 min, filtering, initial filter Liquid discards.Take organic filter of the solution through 0.45 um to filter with 1ml syringes, obtain need testing solution；
Standard solution and need testing solution are each obtained 5 parts.
Chromatographiccondition：Shimadzu LC-20A liquid chromatographs, Waters C18 chromatographic columns（250*4.6,5 um）；Flowing Phase：Acetonitrile：0.5 % phosphoric acid solutions=（20:80）, flow velocity：1.0 ml/min；Sample size：20 ul；Detection wavelength：254 nm. 35 DEG C of column oven.
By above-mentioned chromatographic condition, sample introduction is distinguished to all standard items and test sample, as shown in figure 1,5 samples of standard solution Peak area obtained by product difference sample introduction is 2834815, RSD 0.12%；Peak area average value obtained by 5 samples of need testing solution For 3898300, RSD 0.07%.
According to formula：
Ledermycin zymotic fluid potency：A=A sample * C marks/A mark M sample * n
A samples:The peak area of fermentation broth sample；
A is marked:The peak area of standard items；
C is marked:The potency of standard items（u/ml）；
M samples:The quality of zymotic fluid（g）；
n:The extension rate of zymotic fluid.
Use external standard method standard items and test sample are averagely substituted into can obtain potency that test sample Ledermycins for 11806 u/g。
The methodological study of detection method described in the embodiment of the present invention 1：
（1）Precision Experiment
The same zymotic fluid that Ledermycins is weighed, 6 repetitions is carried out and tests, peak area table 1：
Table 1 Ledermycin zymotic fluid potency repeat experiment area
Number 1 2 3 4 5 6
Peak area 3898300 3887060 3897068 3895181 3886595 3869293
Peak area RSD is 0.28 %, the results showed that, this method detects the zymotic fluid potency precision height that Ledermycins, Favorable reproducibility.
（2）Linear relationship is investigated
According to invention methods described, the reference substance storing solution that potency is 1000 u/ml is prepared with 50ml volumetric flask, is added pure It is respectively 10 u/ml, 20 u/ml, 50 u/ml, 100 u/ml, 150 u/ml, 200 u/ to change water to be diluted to potency concentration respectively Ml, according to this method, to sample concentration（X）And peak area（Y）Carry out data regression, equation of linear regression be Y= 30416X-78136, linear relationship is as shown in figure 3, R2=0.9991, the results showed that zymotic fluid potency of Ledermycining is in 10- In 200 u/ml concentration ranges, peak area and concentration are in good linear relationship.
（3）It is loaded recovery experiment
It is appropriate accurately to weigh the zymotic fluid potency of Ledermycining of known content, then precision adds a certain amount of go respectively Methyl Aureomycin fermentation liquor potency sample 80 %, 100 %, 120 %, make the zymotic fluid effect of Ledermycining in need testing solution Valency is each 3 parts of the high, medium and low region for the zymotic fluid potency standard curve that Ledermycins, according to the operation under item of the present invention, according to Method determines, and calculates the rate of recovery, as a result average recovery rate is 99.46 %, and RSD is 0.58 %, shows that this method average recovery is good.
To sum up show that detection method of content stability of the present invention is good, precision is high, is adapted to fermentation of Ledermycining The measure of liquid potency.
Embodiment 2
Sample and standard items are prepared with embodiment 1.
Chromatographiccondition：Shimadzu LC-20A liquid chromatographs, Waters C18 chromatographic columns（150*4.6,5 um）；Flowing Phase：Acetonitrile：0.5 % phosphoric acid solutions=（30:70）, flow velocity：0.9 ml/min；Sample size：20 ul；Detection wavelength：250 nm. 30 DEG C of column oven.
By above-mentioned chromatographic condition, sample introduction is distinguished to all standard items and test sample, as shown in figure 1,5 samples of standard solution Peak area obtained by product difference sample introduction is 2723203, RSD 0.25%；Peak area average value obtained by 5 samples of need testing solution For 4006298, RSD 0.31%.
According to formula：
Ledermycin zymotic fluid potency：A=A sample * C marks/A mark M sample * n.
Test sample can be obtained by, which averagely being substituted into standard items and test sample using external standard method, Ledermycins zymotic fluid potency For 11109 u/g.
Embodiment 3
Sample and standard items are prepared with embodiment 1.
Chromatographiccondition：Waters C18 chromatographic columns（150*4.6,5 um）；Mobile phase：Acetonitrile：0.5% phosphoric acid solution =（20:80）, flow velocity：0.8 ml/min；Sample size：20 ul；Detection wavelength：260 nm.40 DEG C of column oven.
By above-mentioned chromatographic condition, sample introduction is distinguished to all standard items and test sample, as shown in figure 1,5 samples of standard solution It is that RSD is 0.32 % that product, which distinguish sample introduction gained peak area 2972108,；Peak area average value obtained by 5 samples of need testing solution It is 0.12 % for 3930425, RSD.
According to formula：
Ledermycin zymotic fluid potency：a=ASample*CMark/AMarkMSample*n。
Test sample can be obtained by, which averagely being substituted into standard items and test sample using external standard method, Ledermycins zymotic fluid potency For 11711 u/g.
Embodiment 4
Sample and standard items are prepared with embodiment 1.
Chromatographiccondition：Shimadzu LC-20A liquid chromatographs, Waters C18 chromatographic columns（250*4.6,5 um）；Flowing Phase：Acetonitrile：0.5 % phosphoric acid solutions=（20:80）, flow velocity：1.2 ml/min；Sample size：10 ul；Detection wavelength：260 nm. 40 DEG C of column oven.
By above-mentioned chromatographic condition, sample introduction is distinguished to all standard items and test sample, as shown in figure 1,5 samples of standard solution It is that RSD is 0.32 % that product, which distinguish sample introduction gained peak area 2681747,；Peak area average value obtained by 5 samples of need testing solution It is 0.28 % for 4013300, RSD.
According to formula：
Ledermycin zymotic fluid potency：a=ASample*CMark/AMarkMSample*n。
Test sample can be obtained by, which averagely being substituted into standard items and test sample using external standard method, Ledermycins zymotic fluid potency For 11091 u/g.
It is above-mentioned that a kind of detection method of content of zymotic fluid potency of Ledermycining is carried out with reference to embodiment Detailed description, be illustrative rather than limited, several embodiments can be included according to limited scope.Therefore exist Changing and modifications under present general inventive concept is not departed from, should be belonged within protection scope of the present invention.

Claims (2)

1. a kind of detection method for zymotic fluid potency of Ledermycining, it is characterised in that using high performance liquid chromatography to going first Base Aureomycin fermentation liquor test sample is detected, and then calculates its potency using external standard method；
The chromatographic condition of the high performance liquid chromatography is：
Chromatographic column：C18 posts；
Detector：DAD detectors；
Mobile phase：Acetonitrile：0.5 % phosphoric acid solutions=20-30：80-70；
Flow velocity：0.80-1.20 ml/min；
Sample size：10-20 ul；
Detection wavelength：250-260 nm；
The zymotic fluid that Ledermycins is configured to test sample again after first being dissolved with 0.15 mol/L hydrochloric acid solutions, after dissolving Methyl Aureomycin fermentation liquor potency is in 10-200 u/ml.
2. according to the detection method of the zymotic fluid potency of Ledermycining described in claim 1, it is characterised in that the C18 posts Specification be：The mm of length 250 or 150 mm, the mm of internal diameter 4.6, the um of packing material size 5.
CN201610332799.3A 2016-05-19 2016-05-19 A kind of detection method for zymotic fluid potency of Ledermycining Active CN105738532B (en)

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