CN105277631A - High performance liquid chromatography for detection of residual ceftiofur in pork tissues - Google Patents
High performance liquid chromatography for detection of residual ceftiofur in pork tissues Download PDFInfo
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- CN105277631A CN105277631A CN201410352139.2A CN201410352139A CN105277631A CN 105277631 A CN105277631 A CN 105277631A CN 201410352139 A CN201410352139 A CN 201410352139A CN 105277631 A CN105277631 A CN 105277631A
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Abstract
The invention discloses a high performance liquid chromatography (HPLC) for detection of residual ceftiofur in pork tissues. The ceftiofur relevant residual amount in cattle and pig muscles and kidneys is determined by the high performance liquid chromatography. A sample is extracted by a dithioerythritol solution, is derived by amide, is purified by solid phase extraction columns (C18, SAX and SCX), is separated by 8 column chromatography, and is subjected to gradient elution with acetonitrile-water (with the volume ratio of 15 to 85) containing a 0.1% trifluoroacetic acid as a mobile phase, and is subjected to 6 nm ultraviolet detection. The ceftiofur residual amount shows good linearity with a peak area within 0.016-1.28 mg/L, and the correlation coefficient r is more than 0.9999. The recovery rate with the sample addition concentration of 200-8000 [mu]g/kg is 90%-91%, and the relative standard deviation is 118%-315%. The detection limit of the method (the signal-to-noise ratio S/N is 3) is 50 [mu]g/kg. The method comprises the following steps: 1, instruments and reagents; 2, solution preparation; 3, sample treatment; 4, standard substance treatment; 5, experiment implementation; and 6, data processing.
Description
Technical field
The present invention relates to and a kind ofly detect the residual efficient liquid-phase chromatography method of Ceftiofur in Pork Tissue, belong to field of biological detection.
Background technology
Ceftiofur (ceftiofur) has another name called Ceftlofur, is the third-generation cephalosporin class microbiotic of the 1st animal specific.1988, FDA have approved ceftiofur sodium and is used for the treatment of bovine respiratory bacteriosis, and now this medicine is in U.S.'s Initial Public Offering.After this FDA have approved again ceftiofur sodium, Ceftiofur Hydrochloride and ceftiofur crystalline free acid successively for 1 age in days chicken, turkey, the treatment of the breathing problem of pig, beef cattle, milk cow, horse, goat and sheep etc.Canada, Japan and some countries of Europe also in succession official approval be used for the treatment of breathing problem of pig, beef cattle, milk cow, sheep.Ceftiofur has a broad antifungal spectrum, antibacterial activity is strong, has very strong antibacterial activity to gram positive bacteria, negative bacterium and anaerobion, and to hydrochloric acid in gastric juice and beta lactamase more stable, allergic reaction is few, residual very low in body, is worldwide used widely.At present, domestic You Duo company develops Cefliofur injection in succession, but the dicyandiamide solution of preparation is vegetable oil, soybean wet goods oil medium.First associating Luoyang Huizhong Veterinary Medicine Co. Ltd. of Agricultural University Of South China develops water-based Cefliofur injection at home, because its dicyandiamide solution is water-based, and good fluidity, be easy to absorb, be not easy during intramuscular injection to block syringe needle, little to the pungency of animal, there is wide market application foreground.
At present, in meat tissue, the detection method for Ceftiofur has many, and liquid system is used in conjunction (LC-MS), makings is used in conjunction (GC-MS), ELISA etc.But these method speed are slow, be easily subject to comparatively multifactor impact, accuracy rate is not high.
And high performance liquid chromatography separation efficiency is high, selectivity is good, and detection sensitivity is high, and operation automation analytical precision is high, applied range; The not volatility of test sample and thermal stability restriction, applied range; Mobile phase kind is many, and the optimization by mobile phase reaches high separation efficiency, is therefore more and more taken seriously and adopts.At present also not for the patent of the high performance liquid chromatography of Ceftiofur residue detection.
Summary of the invention
For solving above technical matters, an object of the present invention is that providing a kind of detects the HPLC analytical method that in Pork Tissue, Ceftiofur is residual.
An object of the present invention is achieved in that and detects the residual efficient liquid phase chromatographic analysis instrument of Ceftiofur, and its key is the setting of chromatographic condition and uses cleaning and the activation of front pillar.
The present invention mainly adopts the residual of the Ceftiofur in high efficiency liquid phase chromatographic analysis method detection Pork Tissue, the technology of high-efficient liquid phase analysis method is adopted to mainly contain three aspects, first, the selection of purification condition, extract, derivative after the sample liquid purification solid-phase extraction column that adopts C18 post, SAX anion-exchange column different with SCX cation exchange column 3 kinds.C18 post mainly removes neutral impurity, and SAX post mainly removes cationic compound, and SCX post mainly removes anionic compound.The clean-up effect of 1 post (C18 post), 2 posts (C18 post and SAX post) and 3 posts (C18 post, SAX post and SCX post) has been investigated in this experiment, find to be used alone C18 post and to use the clean-up effect of C18 and SAX post all undesirable, in sample liquid, other component has interference to mensuration.Simultaneously use 3 solid-phase extraction columns, purification and concentration effect better.The second, the selection of mobile phase, mobile phase adopts acetonitrile-water (this system is all containing 0.1%TFA) or methanol-water (containing 0.1%TFA) system, finds that acetonitrile-water system is better than methanol-water solution.Gradient elution is carried out with acetonitrile-water (volume ratio 10: 90) and acetonitrile-water (volume ratio 90: 10), or with acetonitrile-water (volume ratio 15: 85) isocratic elution, the separating effect of Ceftiofur derivant (DCA) is all relatively good, and other components in sample liquid are noiseless to mensuration.Owing to needing balance before gradient elution sample introduction, analysis time is longer, therefore selects isocratic elution.In mobile phase, acetonitrile ratio increases, and Ceftiofur derivant (DCA) goes out peak in advance, but degree of separation is deteriorated.3rd, derivatization: Ceftiofur is main in animal body to be existed with desfuroy leceftiofur (DFC) metabolin, and DFC can combine with biomacromolecule in body (as plasma proteins).DFC in conjunction with state is not easy to be extracted by aqueous solution, therefore will become free state by conjunction with state.Dithioerythritol can be converted into DFC protein bound DFC and parent medicine, and free DFC and iodoacetamide react, and generates the derivant desfuroy leceftiofur acetamide (DCA) with uv absorption.
beneficial effect:detection method has that resolution is high, speed is fast, repeatability is high, automation mechanized operation analytical precision is high, at room temperature just can carry out the advantage such as detecting.
Accompanying drawing explanation
Fig. 1 Ceftiofur standard solution.
Fig. 2 blank muscle HPLC chromatogram.
The blank muscle of Fig. 3 adds the HPLC chromatogram of 1 μ g/g.
Muscle samples HPLC chromatogram after Fig. 4 successive administration.
Embodiment
1. solution preparation:
Ceftiofur storing solution (100mg/L): take 10.0mg Ceftiofur standard items in 100mL volumetric flask, dissolve and constant volume with methyl alcohol ,-20 DEG C of preservations, can use 6 months;
Ceftiofur intermediate liquid (5mg/L): draw 250 μ L100mg/L Ceftiofur storing solutions in 5mL volumetric flask, with 0.025mol/L phosphate buffer (pH7.0) constant volume, shake up (now with the current);
0.025mol/L phosphate buffer (pH7,0)
:3.4g potassium dihydrogen phosphate is dissolved in 900mL water, by 450g/L potassium hydroxide solution adjust ph to 710, is settled to 1L with water;
0.05mol/L borate buffer: take 19g sodium borate and 3.7g potassium chloride, with water constant volume to 1L;
4g/L dithioerythritol solution: take 1.0g dithioerythritol, is dissolved in (now with the current) in 250mL0.05mol/L borate buffer;
40g/L iodoacetamide solution
:take 2.0g iodoacetamide, be dissolved in (now with the current) in 50mL0.025mol/L phosphate buffer.
2. chromatographic condition: C18 post (250mm × 416mm, 5 μm); Mobile phase: acetonitrile-water-trifluoroacetic acid (volume ratio 15: 85: 011); Flow velocity: 110mL/min; Column temperature: 30 DEG C; UV detect wavelength: 266nm; Sample size: 25 μ L.
3. sample preparation:
Extract with derivative
:take 110g tissue sample in 50mL centrifuge tube, add 14mL4g/L dithioerythritol solution, homogeneous, then put into 37 DEG C, constant temperature oscillation tank instrument insulation 20min.Add 3mL4% iodoacetamide solution, shake up, left at room temperature derives 30min, centrifugal 10min (4 DEG C, 20000r/min), takes out supernatant in 25mL color comparison tube.Add 6mL01025mol/L phosphate buffer in residue, mixing, centrifugal 10min (4 DEG C, 20000r/min), merge supernatant, with 25% phosphoric acid solution adjust ph to 2.5 ~ 2.6, be settled to 25mL with water.Take out 10.0mL constant volume liquid, centrifugal 15min (4 DEG C, 20000r/min);
Purification
:the supernatant extracted with derivative last gained is crossed C18 solid-phase extraction column, keep flow velocity 1 ~ 2mL/min, use 5mL phosphate buffer and the drip washing of 3mL0.01moL/L sodium hydroxide solution more successively, be collected in the test tube containing 5mL water with 2mL acetonitrile-water (volume ratio 15: 85) wash-out, keep vacuumizing 2min.Take out the test tube collected, add 5mL water, shake up, cross SAX solid-phase extraction column, keep flow velocity 1 ~ 2mL/min, with 1mL water rinse test tube, add in post, be 5% acetic acid (volume ratio 5: 95) wash-out by 2.5mL acetonitrile-volume fraction and be collected in another containing in the test tube of 5mL water, keeping vacuumizing 2min.Take out the test tube collected, add 5mL water, shake up, cross SCX solid-phase extraction column, keep flow velocity 1 ~ 2mL/min, try with the rinse of 1mL water, add in post, collect with 2.5mL acetonitrile-0.1mol/L sodium chloride (volume ratio 5: 95) wash-out, keep vacuumizing 2min.Take out the test tube collected, shake up, for mensuration;
Standard items process
:pipette appropriate Ceftiofur intermediate liquid to 50mL centrifuge tube, add 14mL4g/L dithioerythritol solution, then put into 37 DEG C, constant temperature oscillation tank instrument insulation 20min.Add 3mL40g/L iodoacetamide solution, shake up, left at room temperature derives 30min, adds 6mL0.025mol/L phosphate buffer, with 25% (volume fraction) phosphoric acid adjust ph to 2.5 ~ 2.6, with water constant volume sample liquid to 25mL.All the other operation stepss are with " purification " operation;
Standard curve making
:accurately take blank homogenised tissue appropriate, add 100 μ L Ceftiofur series standard working fluids, make Ceftiofur concentration in sample be respectively 0.1,0.25,0.5,1,2.5,5,10 μ g/g.By " purification " method process and mensuration, by going the chromatographic peak area of furanylcarbonyl Ceftiofur acetamide (A) to do linear regression with corresponding drug concentration (C), trying to achieve typical curve equation and related coefficient, doing 3 batches altogether;
Detectability and quantitative limit
:accurately take appropriate blank homogenised tissue, interpolation Ceftiofur series standard working fluid in right amount, is configured to serial mark-on sample, by " purification tissue sample pre-treating method " processing sample, then carries out HPLC analysis mensuration by the chromatographic condition of setting.Calculate with concentration limit, mark-on sample concentration during S/N >=3 is detectability, and mark-on sample concentration during S/N >=10 is quantitative limit.
the recovery and precision
This experiment is using pork as sample, getting appropriate standard items joins in 1.0g sample, make addition 200,1000 and 2000 μ g/kg in pork, each Pitch-based sphere respectively gets 24 parts of samples, process by said process, mark-on average recovery rate is 90% ~ 91%, relative standard deviation (RSD) 1.8% ~ 3.5%, and result is satisfied.
application
Get positive pork to carry out processing and measuring according to sample preparation and assay method, in sample, other component is noiseless to mensuration, to quadrature function, obtain J=5.2 × 10 according to processing capacity (being spaced apart 0.5nm) and Origin7.5
-15cm
3lmol
-1; R is obtained again by (2) formula
0value.It is generally acknowledged that medicine mainly comes from 212 trp residues to the fluorescence quenching of BSA.When c (E): c (BSA)=1: 1 (relative molecular mass is in 2000), E=1-I
f/ I
0F=0.13, obtain r value by (1) formula, obtain r=2.15nm.
Claims (4)
1. its feature of high performance liquid chromatography detecting Ceftiofur in Pork Tissue is made up of following part:
Waters highly effective liquid phase chromatographic system, 510 pump housings, 2487 ultraviolet-visible detectors, 717 automatic samplers, join Empowerpro chromatographic software, solid-phase extraction device (Supelco company), C18 solid-phase extraction column (500mg, Varian company), SAX solid-phase extraction column (500mg, Varian company), SCX solid-phase extraction column (100mg, Varian company), reagent: Ceftiofur standard items, dithioerythritol, iodoacetamide, trifluoroacetic acid, acetonitrile, methyl alcohol (chromatographically pure) other reagent are pure for analyzing.
2. a kind ofly according to claim 1 detect the high performance liquid chromatography of Ceftiofur in Pork Tissue and it is characterized in that C18 solid-phase extraction column (500mg, Varian company), with 4mL methyl alcohol and 5mL01025mol/L phosphate buffer drip washing activation before using; SAX solid-phase extraction column (500mg, Varian company), uses 2mL methyl alcohol, 2mL methyl alcohol-011mol/L sodium chloride (volume ratio 25:75) and 2mL water wash to activate successively before using; SCX solid-phase extraction column (100mg, Varian company), uses 1mL methyl alcohol, 2mL methyl alcohol-011moL/L lime chloride (volume ratio 25: 75) and 4mL water wash to activate successively before using.
3. a kind ofly according to claim 2 detect the high performance liquid chromatography of Ceftiofur in Pork Tissue and it is characterized in that methyl alcohol is chromatographically pure, his reagent is pure for analyzing.
4. plant the high performance liquid chromatography detecting Ceftiofur in Pork Tissue according to claim 1 to it is characterized in that as follows
(1) solution preparation:
Ceftiofur storing solution (100mg/L);
Ceftiofur intermediate liquid (5mg/L);
0.025mol/L phosphate buffer (pH7,0);
0.05mol/L borate buffer;
4g/L dithioerythritol solution;
40g/L iodoacetamide solution;
(2) chromatographic condition is set: C18 post (250mm × 416mm, 5 μm); Mobile phase: acetonitrile-water-trifluoroacetic acid (volume ratio 15: 85: 011); Flow velocity: 110mL/min; Column temperature 30 DEG C; UV detect wavelength: 266nm; Sample size: 25 μ L;
(3) sample preparation:
Extract with derive, purify, standard items process.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885854A (en) * | 2017-03-01 | 2017-06-23 | 北京出入境检验检疫局检验检疫技术中心 | The method and its sample-pretreating method of Cephalosporins residual in a kind of detection pluck |
| CN109813667A (en) * | 2017-11-18 | 2019-05-28 | 镇江亿特生物科技发展有限公司 | Ethiprole content uv-spectrophotometric detection method |
| CN110412147A (en) * | 2019-06-12 | 2019-11-05 | 温氏食品集团股份有限公司 | A kind of ceftiofur sodium is in the intracorporal pharmacokinetics of chicken and eliminates detection method |
| CN114113399A (en) * | 2021-12-02 | 2022-03-01 | 天津瑞普生物技术股份有限公司 | Ceftiofur detection method and application thereof |
-
2014
- 2014-07-23 CN CN201410352139.2A patent/CN105277631A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 姜维: "猪、牛的肉和肾脏中头孢噻呋相关残留的分析研究", 《上海海洋大学硕士学位论文》 * |
| 姜维等: "液相色谱法对牛、猪肌肉和肾脏中头孢噻呋残留量的测定研究", 《分析测试学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885854A (en) * | 2017-03-01 | 2017-06-23 | 北京出入境检验检疫局检验检疫技术中心 | The method and its sample-pretreating method of Cephalosporins residual in a kind of detection pluck |
| CN106885854B (en) * | 2017-03-01 | 2019-09-24 | 北京出入境检验检疫局检验检疫技术中心 | The remaining method of Cephalosporins and its sample-pretreating method in a kind of detection pluck |
| CN109813667A (en) * | 2017-11-18 | 2019-05-28 | 镇江亿特生物科技发展有限公司 | Ethiprole content uv-spectrophotometric detection method |
| CN110412147A (en) * | 2019-06-12 | 2019-11-05 | 温氏食品集团股份有限公司 | A kind of ceftiofur sodium is in the intracorporal pharmacokinetics of chicken and eliminates detection method |
| CN114113399A (en) * | 2021-12-02 | 2022-03-01 | 天津瑞普生物技术股份有限公司 | Ceftiofur detection method and application thereof |
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Application publication date: 20160127 |