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		<div class="Basic-Text-Frame">
			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article"><span class="CharOverride-1">Research Article</span></p>
	</div>
		<div class="Basic-Text-Frame">
			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Validation of a High Performance Liquid Chromatography (HPLC) Method for the Determination of Oxytetracycline Residues in Milk</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="ParaOverride-2">&nbsp;</p>
			<p class="ParaOverride-2"><span class="CharOverride-2" lang="en-US">Latifa Boultif</span><span class="CharOverride-3" lang="en-US">1</span><span class="CharOverride-2" lang="en-US">, Noreddine Zeghilet</span><span class="CharOverride-3" lang="en-US">2</span><span class="CharOverride-2" lang="en-US">, Bassem Chebira</span><span class="CharOverride-3" lang="en-US">1</span><span class="CharOverride-2" lang="en-US">, Amir Agabou</span><span class="CharOverride-3" lang="en-US">1</span><span class="CharOverride-2" lang="en-US">, Abdesselam Mekroud</span><span class="CharOverride-3" lang="en-US">1</span><span class="CharOverride-2">&#160;</span><span class="CharOverride-2" lang="en-US">  </span><span class="CharOverride-2"> </span><span class="CharOverride-2">  </span></p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Affiliations"><span class="CharOverride-5">1</span>PADESCA Laboratory, institute of Veterinary science, University of Constantine 1, Algeria; <span class="CharOverride-5">2</span>GSPA Laboratory, institute of Veterinary science, University of Constantine 1, Algeria.</p>
		</div>
		<div>
			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-6">Abstract</span> | Tetracyclines are antibacterial agents widely used in preventing and treating infectious diseases in food producing animals. This extensive use can lead to an increase in their residues levels in food of animal origins. These residues will pass through the food chain to the consumers. In addition, the residues in milk may have many consequences on consumers’ health: allergic reactions in human’s antibiotics give rise to an increase in the antibiotic resistance of pathogenic bacteria, which may result in direct toxity, allergy and resistance to antibiotics. In this way, authors look to detect and quantify Oxytetracycline residues in milk by High Performance Liquid Chromatography (HPLC). They have, at first, proceeded to the optimization of various analysis parameters [stationary phase, mobile phase, absorption spectrum (wavelengths), flow rate and injected volume], then they have continued with their validation using adequate statistical analysis methods (Repetitiveness reproducibility, Linearity of the calibration curve) and the adaptation of an extraction technique of this antibiotic from cow’s milk. The results of the validation process led to the adaptation of following parameters: Sample concentration:&#160;0, 4 mg/mL (diluted in methanol), stationary phase: column C18 reversed phase (Grafted silica gel), mobile phase: acetonitrile, injected volume:&#160;11 µl, wavelength (UV detector):&#160;325 nm and flow rate:&#160;1, 5 mL/min.&#160;The drug was extracted with mixture acetonitrile/HPLC water and simply cleaned up on Whatman filter, then on a nylon membrane filter of 45 µm/ 47 mm.</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-6">Keywords</span> | Oxytetracycline, Residues, Milk, HPLC</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
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		<div class="Basic-Text-Frame">
			<p class="Editor----Citation"><span class="CharOverride-8">Editor</span> | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-6">Received</span> | October 10, 2014; <span class="CharOverride-6">Revised</span> | November 08, 2014; <span class="CharOverride-6">Accepted</span> | November 09, 2014; <span class="CharOverride-6">Published</span> | November 16, 2014&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-6">*Correspondence</span> | Latifa Boultif, University of Constantine 1, Algeria; <span class="CharOverride-6">Email:</span> boultiflatifa@hotmail.fr</p>
			<p class="Editor----Citation"><span class="CharOverride-6">Citation </span>| Boultif L, Zeghilet N, Chebira B, Agabou A, Mekroud A&#160;(2014). Validation of a high performance liquid Chromatography (hplc) method for the determination of oxytetracycline residues in milk. Adv. Anim. Vet. Sci. 2 (10): 574-581.  </p>
			<p class="Editor----Citation"><span class="CharOverride-8">DOI</span> |<a href="http://dx.doi.org/10.14737/journal.aavs/2014/2.10.574.581"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2014/2.10.574.581</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-6" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-6" lang="en-US">(</span><span class="Editor---Citation CharOverride-6" lang="en-US">Online</span><span class="Editor---Citation CharOverride-6" lang="en-US">)</span> |2307-8316; <span class="Editor---Citation CharOverride-6" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-6" lang="en-US">(Print) </span> | 2309-3331</p>
			<p class="Editor----Citation"><span class="CharOverride-8">Copyright </span>© 2014 Boultif et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Heading-1--Introduction----">INTRODUCTION</p>
			<p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">M</span>ilk is an important inexpensive dietary source. It contains valuable proteins and calcium essential for promoting growth in children and general health of the population. In Algeria, milk is considered as the principal source of animal proteins (Amellal, 1995).</p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Tetracyclines produced by Streptomyces <span class="CharOverride-11">spp</span>. are broad spectrum antibiotics active against most Gram-positive and Gram-negative bacteria (Oka et al., 1995). In Algeria, they are used prophylactically and therapeutically in dairy cattle and other domestic animals. However the misuse of these molecules conducts to the presence of their residues in food of animal origin (Wangfuengkanagul et al., 2004).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Antibiotics residues have a negative impact on consumers’ health and food processing industries. Indeed, the presence of antibiotics residues may harm to the consumers’ health through direct toxicity (Guy et al., 2004; Leroy and Fanir, 2005), allergic reactions (Federicci-Mathieu, 2000), carcinogenic processes, pathologies related to the modification of intestinal flora (Broutin et al., 2005; Châtaigner and Stevens, 2005; Fabre and Joyes, 2000), increase of the selection pressure and spread of drug resistant bacteria within animals and human populations (Châtaigner and Stevens, 2005; Sanders, 2005). &#160;For food processing industry, the spoil concerns notably manufacturing of fermented products (Brouillet, 2002; Form, 2003; Maghuin-Rogister et al., 2001). &#160;In fact, the lactic bacteria being sensitive to very low concentrations of antibiotics (Brouillet, 2002; Fabre and Joyes, 2000), the presence of their residues inhibit partially or totally the growth of these ferments and results in many defects (Robb, 2006; Vera, 2005) including accidents in cheese manufacturing, yogurt and other fermented milk products (Broutin et al., 2005; Ryckaert et al., 2003; Zinedine et al., 2007).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In this context, methods must be developed for the monitoring of antibiotics residues notably in milk. &#160;For that two sorts of tests are used: microbiological tests (screening methods) and physicochemical tests (confirmation methods) (Fergusson et al., 2002). &#160;Among this last kink of analyses, High Performance Liquid Chromatography (HPLC)&#160;is recognized as a powerful method for the quantitative analysis of antibiotics residues in milk (Oliveira et al., 2006; Reig and Toldra, 2008). It has several merits; it detects very small amounts of these residues, it presents an extreme sensitiveness and it possesses a great strength of separation and an excellent reproducibility. </p>
		  <p class="Body-Text ParaOverride-1">&#160;</p>
			<p class="Body-Text ParaOverride-1">This work has for principal objective: the detection and the quantification by High Performance Liquid Chromatography of oxytetracycline residues in milk. We optimize various analysis parameters [stationary phase, mobile phase, absorption spectrum (wavelengths), flow rate and injected volume] and the adaptation of an extraction method. &#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Oxytetracycline has been chosen because of its large use in veterinary medicine in Algeria, notably in the treatment of respiratory infections. This antibiotic is also directly implicated in the appearance of dairy industry spoils.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction---- ParaOverride-1"><span class="CharOverride-12">MATERIAL AND METHOD</span></p>
		  <p class="Heading-1--Introduction---- ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Instrumentation </p>
			<p class="Body-Text ParaOverride-1">HPLC Shimadzu corporate Body device consists of : a column (type VP-ODS 250 L*40, filled with grafted silica gel: column C18 in reversed phase), two pumps (models LC-10 ATvp, pumps 1 reference S/N: &#160;C20974009859J2, pump 2 references S/N: &#160;C21014009440CD), two UV detectors &#160;(models SPD-10Avp/10AVvp equipped of a Deuterium lamp, absorption spectrum (UV detector) 190 to 350 nm, reference S/N: &#160;C21004001496LP), a gas cleaner: &#160;(Models DGU-20A5 reference S/N: &#160;L20244301983CR), a control system or integrator (models SCL-10 AVP, reference S/N: &#160;C21014009440CD), an injection loop, two reservoirs of mobile phase and a plastic piping &#160;allowing the progression of the liquid phase in the different compartments of the device. &#160;The other equipment consist of a centrifuge, a filtrating device, cold chamber, analytical scale and HPLC special injection syringes (capacity 20 and 50 µL, reference C 670-12554-03). &#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Reagents</p>
			<p class="Body-Text ParaOverride-1">Methanol ultra-residue of analytical grade (Sigma-Aldrich, Ref. 5363C), Acetonitrile (Biochem. Chemopharma, Ref. 6235A.) and water HPLC rank (Sigma-Aldrich, Ref. 34877).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Liquid chromatography solvents were ﬁltered with 0.45µm Teﬂon and Nylon membranes before used. Puriﬁed samples were ﬁltered through 13mm, 0.45µm Nylon ﬁlters (Supelco, Ref. 58067).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Standards and Work Solutions</p>
			<p class="Body-Text ParaOverride-1">Standard stock solution was prepared by diluting Oxytetracycline hydrochloride (Sigma-Aldrich. Veteranal Ref. 46598) in methanol (4 mg/mL).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The working solutions were prepared daily by appropriately diluting the stock solutions with mobile phase and stored in amber flask at 4°C until use. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Sampling </p>
			<p class="Body-Text ParaOverride-1">Milk sample was collected at a farm located in Constantine (North-East Algeria). &#160;The milk simple is presumed exempt from antibiotics. The collected sample (200 ml) was conveyed quickly in an isothermal tray to the laboratory where it was stored at 4°C. &#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Optimization of Analysis Parameters </p>
			<p class="Body-Text ParaOverride-1">In order to detect and to quantify the oxytetracycline residues in the milk by HPLC, it was necessary to start at first with the optimization of the analysis parameters namely: stationary phase, mobile phase, absorption spectrum (wavelengths), flows rate, injection volume.&#160;This step necessitated more than seven hundred essays. </p>
			<p class="Body-Text ParaOverride-1">&#160;</p>
			<p class="Heading-2--History-in-MM-">Examination of the Method’s Reliability</p>
		  <p class="Heading-2--History-in-MM-">&nbsp; </p>
			<p class="Heading-3">Repetitiveness </p>
			<p class="Body-Text ParaOverride-1">Repetitiveness consists of realizing the technique by the same team at the same laboratory. &#160;The essay results must be obtained by the same operator following the same procedure, using the same equipment and during a current interval of time (According to ISO 3534-1 and ISO 5725-1 standards). To test the Repetitiveness of our obtained results, we carried out three series of 6 essays, on the same day, with the same solution of work, the same operator, the same place, the same equipment and with the same analysis conditions.&#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-3">Reproducibility</p>
			<p class="Body-Text ParaOverride-1">In this case, the essays are realized in different conditions. &#160;At least one of the following parameters is voluntarily modified: operator, device, solution setting, and differed realizations in time (According to ISO 3534-1 and ISO 5725-1 standards). &#160;To achieve this, two different operators carried out three series of twenty essays each, with three different work solutions (one for every series) and in few days apart. </p>
			<p class="Body-Text ParaOverride-1">&#160;</p>
			<p class="Heading-3">Linearity of the Calibration Curve</p>
			<p class="Body-Text ParaOverride-1">The linearity of the variation of the heights peaks area (surfaces) according to the variation of the concentrations of the injected product must be verified for every composite.&#160;The correlation coefficients between heights of the peaks and concentrations, as well as the parameters of the corresponding lines of linear regression must be calculated (ISO 3534-1, ISO 5725-1).&#160;To establish the curve linearity and to determine the regression line for the oxytetracycline, we used different on day prepared dilutions: 1/1, 1/2, 1/4, 1/8, 1/16. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Statistics Study</p>
		  <p class="Heading-2--History-in-MM-">&nbsp;</p>
			<p class="Heading-3">Repeatability Calculation</p>
			<p class="Body-Text ParaOverride-1">We realized an average comparison according to Student‘s<span class="CharOverride-11"> </span>test.&#160;We calculated the average, the standard deviation and the variance from the obtained surfaces to calculate the value of <span class="CharOverride-11">“t”</span> and to compare it with Student’s values at the respective risks of 1% and 5%. &#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The “<span class="CharOverride-11">t</span>” is measured between every two series (<span class="CharOverride-11">a</span> and <span class="CharOverride-11">b</span>, <span class="CharOverride-11">a</span> and <span class="CharOverride-11">c</span>, <span class="CharOverride-11">b</span> and <span class="CharOverride-11">c</span>) according to the following formula: &#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">t = |X<span class="CharOverride-11">a</span> - X<span class="CharOverride-11">b</span>|/√ <span class="CharOverride-15">σ</span>²/N<span class="CharOverride-11">a</span> + <span class="CharOverride-15">σ</span>²/N<span class="CharOverride-11">b</span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">X<span class="CharOverride-11">a</span> being the average of the first series (series a)&#160;</p>
			<p class="Body-Text ParaOverride-1">X<span class="CharOverride-11">b</span> being the average of the second series (series b)</p>
			<p class="Body-Text ParaOverride-1">&#160;<span class="CharOverride-15">σ</span>² being the variance between the two series a and b</p>
			<p class="Body-Text ParaOverride-1">&#160;N<span class="CharOverride-11">a</span> being the number of the repetitions of the series a&#160;</p>
			<p class="Body-Text ParaOverride-1">&#160;N<span class="CharOverride-11">b</span> being the number of the repetitions of the series b&#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">If <span class="CharOverride-11">‘’t’’</span> is inferior to student’s values, the difference between the two series is not significant that means that our method is repeatable or reproducible.&#160;			</p>
			<p class="Body-Text ParaOverride-1">If the calculated <span class="CharOverride-11">‘’t’’</span> is superior to student’s values, the difference between the series is significant and our method is not reliable.&#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-3">Reproducibility Calculation</p>
			<p class="Body-Text ParaOverride-1">The same preceding formula (test of Student) is applied on the obtained surfaces in every series.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-3">Extraction method</p>
			<p class="Body-Text ParaOverride-1">The second section of our study consists in the adaptation of an extraction method of oxytetracycline in milkin this way, we adopted a technique from different recommended methods (Diaz et al., 2007; Helio et al., 2007).&#160;This method is based on the following steps: 10 ml of milk are mixed with 20 ml of acétonitrile and 6 ml of HPLC water. &#160;The mixture is divided up into four tubes, which are homogenized in the vortex for 1 minute then centrifuged at 6000 rpm for 10 min.&#160;Next, the supernatants (aqueous portions) are collected, filtered using Whatman filters.&#160;Then 20 ml of acetonitrile are added to the filtrate, the mixture is divided again into 4 new tubes, which are vortexed during 1 min and centrifuged for the second time at 3000 rpm for 5 min.&#160;Finally, the aqueous portions are harvested and filtered two times: firstly with a Whatman filter, then with a nylon membrane filter of 45 µm/ 47 mm. This method was applied on a milk sample presumed exempt of antibiotics.&#160;A half of the sample quantity was doped with the oxytetracycline standard and the remaining half was used as a control.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"> &#160;</p>
			<p class="Heading-1--Introduction----">RESULTS AND DISCUSSION</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Withheld Parameters&#160;</p>
			<p class="Body-Text ParaOverride-1">Our experimental investigations allowed keeping following HPLC analysis parameters:&#160;Sample conce tration: &#160;0, 4 mg/mL (diluted in methanol), stationary phase: column C18 reversed phase (Grafted silicagel), mobile phase: acetonitrile, injected volume:&#160;11 µl, wavelength (UV detector):&#160;325 nm, flow rate:&#160;1, 5 mL/min and analysis time:&#160;8 min.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The isocratic mode (single mobile phase) was applied during all the experimentation. Various compositions can be used: Wang et al. (2004), Reed et al. (2004); Cinquina et al. (2003) used a mobile phase composed by a combination of acétonitrile and methanol, while for Wan et al. (2013), the mobile phase consisted of acetonitrile and water containing 0.1% formic acid. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Concerning the absorption spectrum, different wavelengths are used. Fritz and Zuo (2007) chose a wavelength of 365 nm, Coyne et al. (2004) used 353 nm, Reed et al. (2004) used 350 nm and Furusawa (2003) used 357 nm.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The flow rate affects the retention time. The increase of flow rate decreases the retention time and vice versa. In many studies a flow rate of 1 ml/min was used (Wang et al., 2004; Cinquina et al., 2003), while in others the chosen flow rate was 1.2 mL/min (Reed et al., 2004).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The figure 1 given below shows the obtained chromatogram while using the withheld parameters, the retention time of the oxytetracycline is 2.15 min.&#160;With other parameters this retention time can change, so for Spisso et al. (2009)&#160;the retention time is 10.80 min. It is 6.13 min for Fritz and Zuo (2007), 4.63 min for Koesukwiwat et al. (2007) and 4.54 min for Zhu et al. (2001).&#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-3">Reliability examination of the withheld parameters&#160;</p>
		  <p class="Heading-3">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">The repeatability</p>
			<p class="Body-Text ParaOverride-1">The obtained results (Table 1), expressed in peak’s area, are subjected to the Student statistical test. The verification of the Repetitiveness is done comparing the series two by two.&#160;After statistical analysis, it is proven that there is no significant difference between the three  series. The <span class="CharOverride-11">“t”</span> in the three cases (table 1) is inferior to the student’s values (at the risks of 1% = 4.6 and at 5% = 2.78). That means that the analysis method is repeatable in the conditions of required analysis.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150111041307.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150111041307.jpg" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/figures/20150111041307.jpg" target="new">Figure 1: </a></b><a href="http://nexusacademicpublishers.com/uploads/figures/20150111041307.jpg"><span>Chromatogram representative of the used parameters</a></p>
          </div>


		  <p class="Heading-2--History-in-MM-">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-">The reproducibility</p>
			<p class="Body-Text ParaOverride-1">The peaks areas (Table 2) are subjected to the statistical tests of Student.&#160;As for the repeatability, it was necessary to compare the series two by two.&#160;We also noticed that there is no significant difference between the three series. Calculated <span class="CharOverride-11">“t” </span>is inferior to Student’s values which confirms that the method is reproducible in our analysis conditions.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">The Linearity of the Calibration Curvwe</p>
			<p class="Body-Text ParaOverride-1">The calibration curve represented in figure 2 is demonstrative. &#160;The correlation coefficient is equal to 0.9830. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">This stage is described in many works as in Koesukwiwat et al. (2007), who reports to have used external standard calibration method. Where the average values were used to constructed calibration curves by plotting the corresponding peak area with analyte concentration. The same operation is made by Sun et al., (2009); Pinar, (2007); Cinquina, (2003).</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6">Table 1:</span> Statistical results of repeatability  p: 6</p>
			<table width="657" height="140" class="Table-Style-1" id="table-1">
				<colgroup>
					<col class="_idGenTableRowColumn-1" />
					<col class="_idGenTableRowColumn-2" />
					<col class="_idGenTableRowColumn-3" />
					<col class="_idGenTableRowColumn-4" />
					<col class="_idGenTableRowColumn-5" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-6">
						<td />
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-20">Series a (n=6)</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-20">Series b (n=6)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-20">Series c  (n=6) </span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Average</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">46134,2</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">45051,3</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">45982,0</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Standard deviation</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">2557,08</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">2748,77</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">2810,47</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Coefficient of variance </span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">7231525494</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">3317104972</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">3285151967</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td rowspan="2">
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Calculated </span><span class="Title CharOverride-21">t</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-21">t</span><span class="Title CharOverride-14"> (a &amp; b)= 0.72</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-21">t</span><span class="Title CharOverride-14"> (b &amp; c)= 0.68</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td colspan="4">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-21">t</span><span class="Title CharOverride-14"> (a&amp;c)= 0.39</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6">Table 2:</span> Statistical results of reproducibility p: 6</p>
			<table width="657" height="163" class="Table-Style-1" id="table-2">
				<colgroup>
					<col class="_idGenTableRowColumn-10" />
					<col class="_idGenTableRowColumn-11" />
					<col class="_idGenTableRowColumn-12" />
					<col class="_idGenTableRowColumn-13" />
					<col class="_idGenTableRowColumn-14" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-9">
						<td />
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-20">Series A (n=20)</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-20">Series B (n=20)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-20">Series C (n=20)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Average</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">41873,8</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">41468,9</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">42206,7</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-15">
						<td>
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Standard deviation</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">1204,7</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">1216,19</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">1243,49</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Variance </span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">1451299,71</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">1479108,05</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">1546257,91</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Confidence Interval</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">41873,8 ± 563.8</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">41468,9 ± 569.2</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-14">42206,7 ± 582.0</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td rowspan="2">
							<p class="Normal" lang="en-US"><span class="Title CharOverride-20">Calculated</span><span class="Title CharOverride-21"> t</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-21">t</span><span class="Title CharOverride-14"> (A&amp;B) = 0.59</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-21">t</span><span class="Title CharOverride-14"> (B&amp;C)= 1.07</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-17">
						<td colspan="4">
							<p class="Normal ParaOverride-3" lang="en-US"><span class="Title CharOverride-21">t</span><span class="Title CharOverride-14"> (A&amp;C)= 0.47</span></p>
						</td>
					</tr>
				</tbody>
			</table>
		  <p>&nbsp;</p>
			<p><br>
			  
			  
		  </p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150111041508.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150111041508.png" width="80" height="80"></a>
  <p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/figures/20150111041508.png" target="new">Figure 2: </a></b><a href="http://nexusacademicpublishers.com/uploads/figures/20150111041508.png"><span>Oxytetracycline calibration curves</a></p>
        </div><br>
          
			
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150111044808.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150111044808.jpg" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/figures/20150111044808.jpg" target="new">Figure 3: </a></b><a href="http://nexusacademicpublishers.com/uploads/figures/20150111044808.jpg"><span>Doped milk sample</a></p>
          </div><br>
          
          <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150111041209.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150111041209.jpg" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/figures/20150111041209.jpg" target="new">Figure 4: </a></b><a href="http://nexusacademicpublishers.com/uploads/figures/20150111041209.jpg"><span>Negative control sample</a></p>
          </div><br>
          
			
			<p class="Heading-2--History-in-MM-">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-">Extraction Method</p>
			<p class="Body-Text ParaOverride-1">In order to eliminate the undesirable components, and to extract the antibiotics in milk, the preparation of the sample is usually necessary. Different methods of extraction can be applied such as: the liquid-liquid extraction, the ultra-filtration, the precipitation of the proteins and the solid phase extraction (Oliveira et al., 2006).&#160;To extract the oxytetracycline from milk, many extraction methods can be used: Fritz and Zuo (2007) proceeded to the centrifugation of sample until the proteins precipitated. Then the solid-phase extraction was used. Zhu et al. (2001) used only Solid-phase extraction (SPE) as extraction method. While Spisso et al. (2009) and Koesukwiwat et al. (2007) used only a liquid extraction. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">After using the adopted extraction method for milk sample, we obtained a transparent and homogenous liquid which is injected directly in the HPLC device. &#160;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">After doping the milk sample by the oxytetracycline standard, we obtained a corresponding peak after 1.90 min (figure 3).&#160;For the negative control sample, no peak could be identified (figure 4).</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">CONCLUSION</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction---- ParaOverride-1"><span class="hps CharOverride-23">This</span><span class="CharOverride-24"> </span><span class="hps CharOverride-23">study involved</span><span class="CharOverride-24"> </span><span class="hps CharOverride-23">the optimization and validation of a High Performance Liquid Chromatography (HPLC) method for the detection and quantification  of oxytetacyclines residues in cow’s milk as well as the adaptation of a</span><span class="CharOverride-24"> </span><span class="hps CharOverride-23">technique for its extraction.</span></p>
			<p class="Heading-1--Introduction---- ParaOverride-1">&nbsp;</p>
			
			
			<p class="Body-Text ParaOverride-1"><span class="hps CharOverride-26">Results in these</span> <span class="hps CharOverride-26">preliminary steps have</span> <span class="hps CharOverride-26">already</span> <span class="hps CharOverride-26">served to detect and quantify</span> <span class="hps CharOverride-26">oxytetracycline residues</span> <span class="hps CharOverride-26">in a</span> <span class="hps CharOverride-26">reduced number of samples.</span> <span class="hps CharOverride-26">This allows us</span> <span class="hps CharOverride-26">to initiate</span> a <span class="hps CharOverride-26">meticulous study on</span> <span class="hps CharOverride-26">the presence</span> <span class="hps CharOverride-26">and quantity of</span> <span class="hps CharOverride-26">this antibiotic</span> <span class="hps CharOverride-26">residues</span> <span class="hps CharOverride-26">in milk</span> <span class="hps CharOverride-26">produced in our region.</span></p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">REFERENCES</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			
			  <li class="References ParaOverride-5" lang="en-US">Amellal R (1995). La filière lait en Algérie: entre objectif de la sécurité alimentaire et la réalité de la dépendance. Options Méditerranéennes. Series B.  14(14): 229-230.</li>
              <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Brouillet P (2002). Résidus de médicaments dans le lait et tests de détection. Bulletin des GVT. 15: 25-41. </li>
				<li class="References ParaOverride-5" lang="en-US">Broutin C (2005). Maîtrise de la qualité dans la transformation laitière. Guide de bonnes pratiques d’hygiène. 29-31.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Châtaigner B, Stevens A (2005). Investigation sur la présence de résidus d’antibiotiques dans les viandes commercialisées à Dakar. Institut Pasteur of Dakar. Pp 6-9.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Cinquina AL, Longo F, Anastasi G, Giannett L, Cozzani R (2003). Validation of a high-performance liquid chromatography method for the determination of oxytetracycline, tetracycline, chlortetracycline and doxycycline in bovine milk and muscle. J. Chromatogr. A. 987(1-2): 227-233. <a href="http://dx.doi.org/10.1016/S0021-9673(02)01446-2"><span class="Hyperlink">http://dx.doi.org/10.1016/S0021-9673(02)01446-2</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Coyne R, Bergh Ø, Samuelsen OB (2004). One-step liquid chromatographic method for the determination of oxytétracycline in fish muscle. J. Chromatogr. B. 810(2): 325-328. <a href="http://dx.doi.org/10.1016/S1570-0232(04)00659-2"><span class="Hyperlink">http://dx.doi.org/10.1016/S1570-0232(04)00659-2</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Diaz TG, Cabanillas AG, Salinas F (1990). Rapid Determination of Sulfathiazole, Oxytetracycline and Tetracycline in Honey by High-Performance Liquid Chromatography. Anal. Lett. 23(4): 607-616. <a href="http://dx.doi.org/10.1080/00032719008052468 "><span class="Hyperlink">http://dx.doi.org/10.1080/00032719008052468</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Fabre JM, Joyes D (2000). Résidus dans le lait: observation des inhibiteurs bien utiliser les médicaments. Proceedings: lait qualité et santé. Pp 10-12.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Federicci-Mathieu C (2000). Résidus dans le lait et sécurité alimentaire: quels risques? quels moyens de maîtrise? Bulletin des GVT. 7: 21-22.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Fergusson JP, Baxter GA, Mac Evoy JD (2002). Detection of streptomycin and dihydrostreptomycin residues in milk, honey and meat. Analyst. 127(7): 951-956. <a href="http://dx.doi.org/10.1039/b200757f"><span class="Hyperlink">http://dx.doi.org/10.1039/b200757f</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Form G (2003). Les résidus inhibiteurs dans le lait. Evolution des méthodes de détection Facteurs de risques en région Rhône-Alpes. These of veterinary medecin. University Claude-Bernard-Lyon. Pp 26-30.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Fritz JW, Zuo Y (2007). Simultaneous determination of tetracycline, oxytetracycline, and 4-epitetracycline in milk by high-performance liquid chromatography. Food Chem. 105(3): 1297-1301. <a href="http://dx.doi.org/10.1016/j.foodchem.2007.03.047"><span class="Hyperlink">http://dx.doi.org/10.1016/j.foodchem.2007.03.047</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Furusawa N (2003). Isolation of tetracyclines in milk using a solid-phase extracting column and water eluent. Talanta. 59(1): 155-159. <a href="http://dx.doi.org/10.1016/S0039-9140(02)00472-1"><span class="Hyperlink">http://dx.doi.org/10.1016/S0039-9140(02)00472-1</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Guy PA, Royer D, Mottier P, Gremaud E, Perisset A, Stadler RH (2004). Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry. J. Chromatogr. A. 1054(1-2): 365-371. <a href="http://dx.doi.org/10.1016/S0021-9673(04)01290-7"><span class="Hyperlink">http://dx.doi.org/10.1016/S0021-9673(04)01290-7</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Helio A, Martins J, Tereza A, Kussumi A, Wang D, Lebre T (2007). A rapid method to determine antibiotic residues in milk using liquid chromatography coupled to electrospray tandem mass spectrometry. J. Braz. Chem. Soc. 18(2): 52-71.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Koesukwiwat U, Jayanta S, Leepipatpiboon N (2007). Validation of a liquid chromatography–mass spectrometry multi-residue method for the simultaneous determination of sulfonamides, tetracyclines, and pyrimethamine in milk. J.Chromatogr. A. 1140(1-2): 147-156. <a href="http://dx.doi.org/10.1016/j.chroma.2006.11.099"><span class="Hyperlink">http://dx.doi.org/10.1016/j.chroma.2006.11.099</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Leroy P, Fanir F (2005). Méthode statistique en médecine vétérinaire. Veterinary Faculty of Liege. Pp 49-133.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Maghuin-Rogister G, Janosi A, Helbo V, Van Peteghem C, Sander E, Van Eeckhout E, Cornelis M, Jouret M (2001). Stratégie intégrée d’analyse qualitative et quantitative des résidus de substances antimicrobiennes dans les denrées alimentaires. Services scientifiques du premier Ministre Affaires scientifiques, techniques et culturelles (SSTC) France. Rapport Final SSTC. Pp 13-58.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Oka H, Patterson J, Nakazawa H, Harada KI, Macneil JD (1995). Chemical Analysis for Antibiotics Used in Agriculture. AOACINT. Arlington USA. Pp. 333.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Oliveira R, De Pietro A, Cass Q (2006). Quantification of cephalexin as residue levels in bovine milk by high-performance liquid chromatography with on-line sample cleanup. Talanta. 71(3): 1233-1238. <a href="http://dx.doi.org/10.1016/j.talanta.2006.06.024"><span class="Hyperlink">http://dx.doi.org/10.1016/j.talanta.2006.06.024</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Pınar A (2007). The confirmation of the commercial kits used in the detection of antibiotics in milk with HPLC (High Pressure Liquid Chromatography), master of science In food engineering, school of Engineering and Sciences of İzmir. Turkey. Pp. 10-44.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Reed L A, Siewicki T C, Shah J C (2004). Pharmacokinetics of oxytétracycline in the white shrimp, Litopenaeus setiferus. Aquaculture. 232(1-4): 11-28. <a href="http://dx.doi.org/10.1016/S0044-8486(03)00451-4"><span class="Hyperlink">http://dx.doi.org/10.1016/S0044-8486(03)00451-4</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Reig M, Toldra F (2008). Veterinary drug residues in meat: concerns and rapid methods for detection. Meat Sci. 78(1-2): 60-67. <a href="http://dx.doi.org/10.1016/j.meatsci.2007.07.029"><span class="Hyperlink">http://dx.doi.org/10.1016/j.meatsci.2007.07.029</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Robb ED (2006). Pour un fromage de meilleure qualité. Revue: pour l’amour des vaches. 5(1): 6.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Ryckaert (2003). questions sur le lait. Édition IMP Bruxelles. 13-56.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Sanders P (2005). L’antibiorésistance en médecine vétérinaire: enjeux de santé publique et de santé animale. Revue: Bulletin de l’académie vétérinaire de France. 158(2): 139-140.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Spisso BF, Gonc de Araúcho MA, Monteiro MA, Belem Lima AM, Pereira MU, Luiz RA, Nóbrega AW (2009). A liquid chromatography–tandem mass spectrometry conﬁrmatory assay for the simultaneous determination of several tetracyclines in milk considering keto-enol tautomerism and epimerization phenomena. Anal. Chim. Acta. 656(1-2): 72-84. <a href="http://dx.doi.org/10.1016/j.aca.2009.10.012"><span class="Hyperlink">http://dx.doi.org/10.1016/j.aca.2009.10.012</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Sun X, He X, Zhang Y, Chen L (2009). Determination of tetracyclines in food samples by molecularly imprinted monolithic column coupling with high performance liquid chromatography. Talanta. 79(3): 926-934. <a href="http://dx.doi.org/10.1016/j.talanta.2009.05.033"><span class="Hyperlink">http://dx.doi.org/10.1016/j.talanta.2009.05.033</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Vera G (2005). La rationalisation des traitements des mammites en exploitation laitière. These of veterinary medecin. National school of Alfort. Pp 26-40.</li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Wan Y, Jia A, Zhu Z, Hu J (2013). Transformation of tetracycline during chloramination: Kinetics, products and pathways. Chemosphere. 90(4): 1427-1434. <a href="http://dx.doi.org/10.1016/j.chemosphere.2012.09.001"><span class="Hyperlink">http://dx.doi.org/10.1016/j.chemosphere.2012.09.001</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Wang Q, Liu Q, Li J (2004). Tissue distribution and elimination of oxytétracycline in perch Lateolabras janopicus and black seabream (Sparus macrocephalus) following oral administration. Aquaculture. 237: 31-40. <a href="http://dx.doi.org/10.1016/j.aquaculture.2004.03.016"><span class="Hyperlink">http://dx.doi.org/10.1016/j.aquaculture.2004.03.016</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Wangfuengkanagul N, Siangproh W, Chailapakul O (2004). A flow injection method for the analysis of tetracycline antibiotics in pharmaceutical formulations using electrochemical detection at anodized boron-doped diamond thin film electrode. Talanta. 64(5): 1183-1188. <a href="http://dx.doi.org/10.1016/j.talanta.2004.04.032"><span class="Hyperlink">http://dx.doi.org/10.1016/j.talanta.2004.04.032</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Zhu J, Snow DD, Cassada DA, Monson SJ, Spalding RF (2001). Analysis of oxytetracycline, tetracycline, and chlortetracycline in water using solid-phase extraction and liquid chromatography and tandem mass spectrometry. J. Chromatogr. A. 928(2): 177-186. <a href="http://dx.doi.org/10.1016/S0021-9673(01)01139-6"><span class="Hyperlink">http://dx.doi.org/10.1016/S0021-9673(01)01139-6</span></a></li>
                <p>&nbsp;</p>
				<li class="References ParaOverride-5" lang="en-US">Zinedine A, Faid M, Benlemlih M (2007). Detection of antibiotics residues in the milk and in dairy products by microbiological method. Remise. 1(1): 1-9.</li>
                <p>&nbsp;</p>
                <p>&nbsp;</p>
          <p>&nbsp;</p>
			
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