CN104330497A - Method for determining fluoroquinolone drug residual in beef - Google Patents
Method for determining fluoroquinolone drug residual in beef Download PDFInfo
- Publication number
- CN104330497A CN104330497A CN201410666699.5A CN201410666699A CN104330497A CN 104330497 A CN104330497 A CN 104330497A CN 201410666699 A CN201410666699 A CN 201410666699A CN 104330497 A CN104330497 A CN 104330497A
- Authority
- CN
- China
- Prior art keywords
- beef
- extraction
- ultrasonic
- extract
- assay method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for determining fluoroquinolone drug residual in beef. The method comprises the following steps: accurately weighing a beef test sample, adding an ultrasonic extracting solvent for extracting, centrifuging the extract, purifying the obtained centrifuged liquid supernatant by use of a reverse solid-phase extraction column, next, eluting by use of 5mL of methanol, drying the obtained eluate by use of a nitrogen blowing instrument at the temperature of 60 DEG C, dissolving the dried eluate in a moving phase, filtering the solution by use of a 0.22-micron filter membrane and putting the filtrate into a sample injection bottle, and finally, determining the filtrate by use of a high performance liquid chromatograph with a fluorescence detector. The method for determining the fluoroquinolone drug residual in the beef has the beneficial effects that two main factors, namely the extraction time and the extract dosage, are optimized so that the optimal ultrasonic extraction time and the optimal extract material ratio can be obtained, and the method has the advantages of good extraction effect, short time consumption, good sensitivity, precision and accuracy and the like, and is suitable for quick detection on the fluoroquinolone drug residual in the beef samples.
Description
Technical field
The present invention relates to the assay method of meat products medicament residue, be specifically related to the assay method of fluorine promise quinoline ketone medicament residue in beef.
Background technology
Fluoquinolone is a class antimicrobial, and mechanism of action is: anti-bacteria DNA helicase, hinders DNA replication dna.Fluoroquinolones is to G-bacillus, and comprising Pseudomonas aeruginosa all has good antibacterial action, to the certain antibacterial activity of G+ coccus also tool, especially to resistance G-bacillus, still can present sensitivity.Clinically be used for the treatment of the infection such as urinary tract, enteron aisle, respiratory tract and skin soft tissue, abdominal cavity, Bones and joints.Any medicine has spinoff, fluoquinolone is no exception, and due to the widespread use of fluoroquinolones, Irrational Use of Drugs and drug abuse situation is usually had to occur, the bad reaction of flouroquinolone drugs mainly occurs in the mankind, animal then reacts little, but the medicament residue after animal-use drug can be propagated by food chain, the generation of mankind's bad reaction phenomenon can be caused equally, affect human health, for this reason, the residue detection of fluoquinolone in animal body is more and more caused to the attention of people.
Ultrasound wave extraction and isolation is mainly according to a subject of the design such as existence, polarity, dissolubility of effective constituent in material and effective constituent colony.The method of Appropriate application ultrasound wave vibration carries out the new technology extracted, and solvent is entered in solid matter rapidly, is as far as possible fully dissolved among solvent by the organic principle contained by its material, obtains multicomponent mixed extract.Utilize ultrasonic technology to strengthen extraction and isolation process, can effectively improve extraction and isolation rate, shorten extraction time, cost-saving, the Quality and yield that even can also improve product.The unique advantage that ultrasound wave extraction (also referred to as ultrasonic extraction) is low with its Extracting temperature, extraction ratio is high, extraction time is short is applied to the extraction of Chinese crude drug and various animal and plant effective content by having creativity consciousness person, be to substitute tradition to shear the modern high technology means that process realizes efficiently, energy-saving and environmental protection formula is extracted.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, provides the assay method of fluorine promise quinoline ketone medicament residue in a kind of beef.
To achieve these goals, technical scheme provided by the invention is: the assay method of fluorine promise quinoline ketone medicament residue in beef, comprises the following steps:
1) beef sample 1.99-2.01g is accurately taken, put into the tool plug ground conical flask of 50mL, the ratio adding 2.5mL-12.5mL according to every 1g beef sample adds ultrasonic extraction solvent, covers tightly stopper and is placed on ultrasonic cleaning instrument and extracts, extraction time is 5-25min, is extracted liquid;
2) extract after extraction step 1) obtained is centrifugal immediately, and centrifugal rotational speed is 8000r/m, and centrifugation time 6min, obtains centrifuged supernatant;
3) by step 2) centrifuged supernatant that obtains crosses reverse phase solid phase extraction column purification, then with 5mL methanol-eluted fractions, obtains eluent;
4) eluent step 3) obtained dries up with Nitrogen evaporator under temperature 60 C, then after dissolving with mobile phase, with 0.22 μm of membrane filtration in sample injection bottle, on carry out measuring with the high performance liquid chromatograph of fluorescence detector.
Further, the assay method of fluorine promise quinoline ketone medicament residue in above-mentioned beef, in described step 1), beef sample weight is 2g; Ultrasonic extraction solvent is according to volume basis acetonitrile: 10% solution of trichloroacetic acid is 30:70; The additional proportion of described ultrasonic extraction solvent is that every 1g beef sample adds 7.5mL ultrasonic extraction solvent; Described extraction time is 20min.
Further, the assay method of fluorine promise quinoline ketone medicament residue in above-mentioned beef, in described step 3), reverse phase solid phase extraction post need first activate with 3mL methyl alcohol and 3mL water successively.
Further, the assay method of fluorine promise quinoline ketone medicament residue in above-mentioned beef, in described step 4), mobile phase is according to volume basis citric acid/ammonium acetate buffer: acetonitrile is 82:18, flow velocity is 0.8mL/min, and column temperature is 25 DEG C, and sampling volume is 20 μ L, fluorescence detector excitation wavelength is 280nm, and emission wavelength is 450nm.
Beneficial effect of the present invention is: the assay method of fluorine promise quinoline ketone medicament residue in beef provided by the invention, ultrasonic extraction technology is applied in the middle of the residual detection of fluorine quinolone druge in beef, ultrasonic extraction technology is adopted to extract residual fluorine quinolone druge in the middle of beef, and extraction time and extract consumption two topmost factors are optimized, obtain best ultrasonic extraction time and extract material ratio, the sample of ultrasonic extraction is after Solid phase extraction, measure with the high performance liquid chromatograph with fluorescence detector, it is good that the method has extraction effect, consuming time short, sensitivity, the advantages such as preci-sion and accuracy is good, be suitable for the quick detection of fluo quinolone drug residual in beef sample.The recovery of the method is between 62-97%, and relative standard deviation RSD < 15%, minimum detectability is between 4.05-13.3 μ g/kg.
Accompanying drawing explanation
Fig. 1 is the chromatogram of beef mark-on 25 μ g/kg.
Embodiment
agents useful for same of the present invention and instrument:
Instrument:
KQ-600DE type ultrasonic cleaning instrument: Kunshan ultrasonic cleaning instrument plant;
XS205 type electronic balance: Mettler Toledo company;
Homogenate instrument: waters company of the U.S.;
The refined hydro-extractor in H-2050R type Hunan;
Nitrogen evaporator: waters company of the U.S.;
Waters 2695 type high performance liquid chromatograph (being furnished with fluorescence detector): waters company of the U.S.;
SunFireTMC18 post: 4.6 mm × 250 mm, 5 μm, waters company of the U.S..
Reagent:
Citric acid, ammonium acetate, trichloroacetic acid and NaOH are analytical reagent: purchased from Shanghai traditional Chinese medicines group;
Methyl alcohol, acetonitrile: chromatographically pure, purchased from Belgian fisher company;
Experimental water is ultrapure water;
Ciprofloxacin, Enrofloxacin, Danofloxacin and sarafloxacin standard items: purchased from German Dr.Ehrenstorfer company.
Citric acid/ammonium acetate buffer liquid and preparation method thereof: take 10.6g citric acid and 7.9g ammonium acetate, with pure water constant volume to 1mL, drips triethylamine 1mL, crosses 0.22 μm of filter membrane for subsequent use.
Ultrasonic extract preparation method: volume ratio is acetonitrile-10% solution of trichloroacetic acid of 30:70, measures 300 mL acetonitriles and 700 mL 10% solution of trichloroacetic acid dissolve each other.
embodiment 1:
1, ultrasonic extraction:
Accurately take sample 2.0 ± 0.01 g, put into the tool plug ground conical flask of 50mL, adding a certain amount of extract is extract, cover tightly stopper, be placed on ultrasonic cleaning instrument and extract, after extraction, extract is centrifugal immediately, solid phase extraction column purification crossed by supernatant, methanol-eluted fractions, eluent dries up with Nitrogen evaporator at 60 DEG C.Finally dissolve with mobile phase, be filled into inside sample injection bottle, upper high performance liquid chromatograph measures.
2, liquid chromatography instrument method:
Mobile phase is citric acid/ammonium acetate buffer-acetonitrile (V:V=82:18), and flow velocity is 1.0mL/min, column temperature 25 DEG C, sampling volume 20 μ l, fluorescence detector excitation wavelength: 280nm, emission wavelength 450nm.
3, the optimization of ultrasonic extraction conditions:
Fluoroquinolones has similar chemical property and physical property, therefore select Ciprofloxacin to be representative, adopt the husky star standard solution of ringing third (25 μ g/kg) in beef sample, after treatment, measure Ciprofloxacin chromatographic peak peak area, peak area can react best extraction conditions.
1) extraction time:
Other conditions are constant, to set ultrasonic extraction time be respectively 5min, 10min, 15min, 20min and 25min is single factor test condition, measure the chromatographic peak peak area of the mark-on Ciprofloxacin beef sample under different ultrasonic extraction time, ultrasonic extraction extraction time on extraction effect to affect result as shown in table 1.Along with the prolongation of ultrasonic extraction time, in sample, the peak area that detects of Ciprofloxacin increases gradually; After 20 min, gradually stable.Prolongation along with ultrasonic time can cause the loss of Avermectins medicine, and therefore 15 min are the best ultrasonic extraction time.
Table 1
。
2) consumption of ultrasonic extraction solvent:
Acetonitrile-10% solution of trichloroacetic acid that use volume ratio is 30:70 is as ultrasonic extraction solvent.When other conditions are constant, when sampling amount is 2g, add extract be respectively 5,10,15,20,25mL, ultrasonic extraction Solvent quantity on extraction effect to affect result as shown in table 2.Found that when extraction solvent consumption is 15mL, effect of extracting is best.When extraction solvent consumption is less, residue of ciprofloxacin extraction not exclusively, has part to remain in meat; When solvent load is too large, effect of extracting is not affected, but cause the waste of solvent.
Table 2
。
4. best ultrasonic extraction conditions:
By above-mentioned single factor test Optimal Experimental, show that best extraction conditions is as follows: add extraction agent according to the ratio of lg/7.5 mL, ultrasonic extraction 20min.The effect of extracting of Avermectins medicine is best under these conditions.
embodiment 2:
The assay method of fluorine promise quinoline ketone medicament residue in beef, comprises the following steps:
1) beef sample 1.99g is accurately taken, put into the tool plug ground conical flask of 50mL, the ratio adding 2.5mL according to every 1g beef sample adds ultrasonic extraction solvent, cover tightly stopper to be placed on ultrasonic cleaning instrument and to extract, extraction time is 5min, be extracted liquid, acetonitrile-10% solution of trichloroacetic acid of ultrasonic extraction solvent to be volume ratio be 30:70;
2) extract after extraction step 1) obtained is centrifugal immediately, and centrifugal rotational speed is 8000r/m, and centrifugation time 6min, obtains centrifuged supernatant;
3) by step 2) centrifuged supernatant that obtains crosses reverse phase solid phase extraction column purification, then with 5ml methanol-eluted fractions, obtains eluent; Reverse phase solid phase extraction post need first activate with 3ml methyl alcohol and 3ml water successively;
4) eluent step 3) obtained dries up with Nitrogen evaporator under temperature 60 C, after dissolving with mobile phase again, with 0.22 μm of membrane filtration in sample injection bottle, on carry out measuring with the high performance liquid chromatograph of fluorescence detector, the citric acid/ammonium acetate buffer-acetonitrile of mobile phase to be volume ratio be 82:18, flow velocity is 0.8ml/min, column temperature is 25 DEG C, sampling volume is 20 μ l, and fluorescence detector excitation wavelength is 280nm, and emission wavelength is 450nm.
embodiment 3:
The assay method of fluorine promise quinoline ketone medicament residue in beef, comprises the following steps:
1) beef sample 2.01g is accurately taken, put into the tool plug ground conical flask of 50mL, the ratio adding 12.5mL according to every 1g beef sample adds ultrasonic extraction solvent, cover tightly stopper to be placed on ultrasonic cleaning instrument and to extract, extraction time is 25min, be extracted liquid, acetonitrile-10% solution of trichloroacetic acid of ultrasonic extraction solvent to be volume ratio be 30:70;
2) extract after extraction step 1) obtained is centrifugal immediately, and centrifugal rotational speed is 8000r/m, and centrifugation time 6min, obtains centrifuged supernatant;
3) by step 2) centrifuged supernatant that obtains crosses reverse phase solid phase extraction column purification, then with 5ml methanol-eluted fractions, obtains eluent; Reverse phase solid phase extraction post need first activate with 3ml methyl alcohol and 3ml water successively;
4) eluent step 3) obtained dries up with Nitrogen evaporator under temperature 60 C, after dissolving with mobile phase again, with 0.22 μm of membrane filtration in sample injection bottle, on carry out measuring with the high performance liquid chromatograph of fluorescence detector, the citric acid/ammonium acetate buffer-acetonitrile of mobile phase to be volume ratio be 82:18, flow velocity is 0.8ml/min, column temperature is 25 DEG C, sampling volume is 20 μ l, and fluorescence detector excitation wavelength is 280nm, and emission wavelength is 450nm.
embodiment 4:
The assay method of fluorine promise quinoline ketone medicament residue in beef, comprises the following steps:
1) beef sample 2g is accurately taken, put into the tool plug ground conical flask of 50mL, the ratio adding 7.5mL according to every 1g beef sample adds ultrasonic extraction solvent, cover tightly stopper to be placed on ultrasonic cleaning instrument and to extract, extraction time is 20min, be extracted liquid, acetonitrile-10% solution of trichloroacetic acid of ultrasonic extraction solvent to be volume ratio be 30:70;
2) extract after extraction step 1) obtained is centrifugal immediately, and centrifugal rotational speed is 8000r/m, and centrifugation time 6min, obtains centrifuged supernatant;
3) by step 2) centrifuged supernatant that obtains crosses reverse phase solid phase extraction column purification, then with 5ml methanol-eluted fractions, obtains eluent; Reverse phase solid phase extraction post need first activate with 3ml methyl alcohol and 3ml water successively;
4) eluent step 3) obtained dries up with Nitrogen evaporator under temperature 60 C, after dissolving with mobile phase again, with 0.22 μm of membrane filtration in sample injection bottle, on carry out measuring with the high performance liquid chromatograph of fluorescence detector, the citric acid/ammonium acetate buffer-acetonitrile of mobile phase to be volume ratio be 82:18, flow velocity is 0.8ml/min, column temperature is 25 DEG C, sampling volume is 20 μ l, and fluorescence detector excitation wavelength is 280nm, and emission wavelength is 450nm.Chromatographic peak peak sequence is Ciprofloxacin, Danofloxacin, Enrofloxacin and sarafloxacin successively.As shown in Figure 1, utilize typical curve external standard method, just can to measure in Ciprofloxacin, Danofloxacin, Enrofloxacin and sarafloxacin the content of one or more.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. the assay method of fluorine promise quinoline ketone medicament residue in beef, is characterized in that, comprise the following steps:
1) beef sample 1.99-2.01g is accurately taken, put into the tool plug ground conical flask of 50mL, the ratio adding 2.5mL-12.5mL according to every 1g beef sample adds ultrasonic extraction solvent, covers tightly stopper and is placed on ultrasonic cleaning instrument and extracts, extraction time is 5-25min, is extracted liquid;
2) extract after extraction step 1) obtained is centrifugal immediately, and centrifugal rotational speed is 8000r/m, and centrifugation time 6min, obtains centrifuged supernatant;
3) by step 2) centrifuged supernatant that obtains crosses reverse phase solid phase extraction column purification, then with 5ml methanol-eluted fractions, obtains eluent;
4) eluent step 3) obtained dries up with Nitrogen evaporator under temperature 60 C, then after dissolving with mobile phase, with 0.22 μm of membrane filtration in sample injection bottle, on carry out measuring with the high performance liquid chromatograph of fluorescence detector.
2. the assay method of fluorine promise quinoline ketone medicament residue in beef according to claim 1, it is characterized in that, in described step 1), beef sample weight is 2g; Ultrasonic extraction solvent is according to volume basis acetonitrile: 10% solution of trichloroacetic acid is 30:70; The additional proportion of described ultrasonic extraction solvent is that every 1g beef sample adds 7.5mL ultrasonic extraction solvent; Described extraction time is 20min.
3. the assay method of fluorine promise quinoline ketone medicament residue in beef according to claim 2, it is characterized in that, in described step 3), reverse phase solid phase extraction post need first activate with 3ml methyl alcohol and 3ml water successively.
4. the assay method of fluorine promise quinoline ketone medicament residue in beef according to claim 3, it is characterized in that, in described step 4), mobile phase is according to volume basis citric acid/ammonium acetate buffer: acetonitrile is 82:18, flow velocity is 0.8mL/min, and column temperature is 25 DEG C, and sampling volume is 20 μ L, fluorescence detector excitation wavelength is 280nm, and emission wavelength is 450nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410666699.5A CN104330497A (en) | 2014-11-20 | 2014-11-20 | Method for determining fluoroquinolone drug residual in beef |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410666699.5A CN104330497A (en) | 2014-11-20 | 2014-11-20 | Method for determining fluoroquinolone drug residual in beef |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104330497A true CN104330497A (en) | 2015-02-04 |
Family
ID=52405265
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410666699.5A Pending CN104330497A (en) | 2014-11-20 | 2014-11-20 | Method for determining fluoroquinolone drug residual in beef |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104330497A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105403522A (en) * | 2015-10-26 | 2016-03-16 | 南昌大学 | Method for rapidly detecting fluoroquinolone residual in foods |
| CN106526099A (en) * | 2016-11-09 | 2017-03-22 | 无锡艾科瑞思产品设计与研究有限公司 | Method for detecting quinolones in food |
| CN109738559A (en) * | 2019-03-06 | 2019-05-10 | 烟台大学 | Methanol saltout split-phase extract meat products in quinolones residual pre-treating method |
| CN110221008A (en) * | 2019-06-25 | 2019-09-10 | 华中农业大学 | A method of Danofloxacin mesylate in detection Swine plasma and alveolar fluid |
-
2014
- 2014-11-20 CN CN201410666699.5A patent/CN104330497A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| 刘靖靖等: "高效液相色谱法同时检测8种喹诺酮类兽药残留量", 《分析试验室》 * |
| 李娟: "动物组织中11种喹诺酮类药物多残留的HPLC法研究", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
| 杜振霞等: "牛肉中三种氟喹诺酮类兽药残留的UPLC-MS/MS分析方法", 《质谱学报》 * |
| 林保银: "鸡肉中11种喹诺酮类药物多残留的高效液相色谱检测", 《色谱》 * |
| 顾慧儿等: "诺氟沙星的HPLC测定", 《中国医药工业杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105403522A (en) * | 2015-10-26 | 2016-03-16 | 南昌大学 | Method for rapidly detecting fluoroquinolone residual in foods |
| CN105403522B (en) * | 2015-10-26 | 2018-11-02 | 南昌大学 | A kind of quickly detection remaining method of Fluorine in Foods quinolones |
| CN106526099A (en) * | 2016-11-09 | 2017-03-22 | 无锡艾科瑞思产品设计与研究有限公司 | Method for detecting quinolones in food |
| CN109738559A (en) * | 2019-03-06 | 2019-05-10 | 烟台大学 | Methanol saltout split-phase extract meat products in quinolones residual pre-treating method |
| CN110221008A (en) * | 2019-06-25 | 2019-09-10 | 华中农业大学 | A method of Danofloxacin mesylate in detection Swine plasma and alveolar fluid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104330497A (en) | Method for determining fluoroquinolone drug residual in beef | |
| CN102406749B (en) | HPLC (high-performance liquid chromatography) detection method capable of simultaneously detecting contents of six amino acids in donkey-hide glue blood-supplementing preparation | |
| Hutsell et al. | Determination of disopyramide and its mono-N-dealkylated metabolite in blood serum and urine | |
| CN109374764A (en) | Eight taste dragons of one kind bore particle HPLC finger-print and Contents of Main Components measuring method | |
| CN104215709A (en) | Method for determining residual abamectin antibiotics in beef | |
| CN103235117A (en) | Beta 2-acceptor stimulant ELISA kit and usage method and application thereof | |
| CN107315058A (en) | A kind of method of total ginkgoic acid in detection ginkgo biloba succi | |
| CN109959735A (en) | The content assaying method of three kinds of ingredients in line leaf broom top, line leaf goldspink flower extract and line leaf goldspink jasmine tea | |
| CN105203673B (en) | The high-efficiency liquid chromatography method for detecting of Compound Solanum Nigrum sulfathiazole | |
| CN105301123A (en) | HPLC detection method for alpinia-cyperus preparations | |
| WO2024007538A1 (en) | Content measurement method for six alkaloid components in small meridian-activating pill | |
| CN105929066A (en) | Method for determining andrographolide and dehydroandrographoline in andrographis tablet by using HPLC | |
| CN104345117A (en) | Qualitative and quantitative detection method of compound Japanese Ardisia Herb tablets | |
| CN103513000B (en) | Method for carrying out identification and content measurement on nymphaea candida presl | |
| CN103267824B (en) | Method for detecting wind-cold cold granules | |
| Browning Jr et al. | Gas chromatographic determination of hexachlorophene in blood and urine | |
| CN106198821B (en) | A kind of method that sulfa antibiotics remain in detection milk | |
| Zhao et al. | Use of cloud point extraction with derivatizing reagent for the extraction and determination of isoniazid | |
| CN104483411B (en) | A kind of capsule of weeping forsythia and the detection method containing capsule of weeping forsythia product | |
| CN103869020B (en) | A kind of method differentiating natural musk and muscone | |
| CN103529141B (en) | The content assaying method of calculus bovis factitius mesobilirubin after a kind of inclusion | |
| CN101306126B (en) | Quality control method of compound south isatis root granules | |
| CN107782830A (en) | The content assaying method of Isopsoralen in a kind of oteoporosis treating medicinal capsule | |
| CN101596229A (en) | The method of quality control of Ramulus Et Folium Pithecellobii Lucidi extract and preparation thereof | |
| CN110618220A (en) | Method for detecting content of aristolochic acid I in Shangtongning capsules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150204 |
|
| RJ01 | Rejection of invention patent application after publication |