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<body id="AAVS_MH20150227090208_-Chebira-et-al" xml:lang="en-GB">
		<div class="Basic-Text-Frame">
		  <p class="Type-of-Article"><span class="CharOverride-1">Research Article</span></p>
		  <p class="Type-of-Article">&nbsp;</p>
	</div>
		<div class="Basic-Text-Frame">
		  <p class="title- ParaOverride-1">Validation of a High Performance Liquid Chromatography (HPLC) Method to Detect and Quantify Some Antibiotics Residues in Honey</p>
		  <p class="title- ParaOverride-1">&nbsp;</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Authors"><span class="CharOverride-2">Bassem Chebira*, Latifa Boultif, Amir Agabou, Abdesselam Mekroud</span></p>
		</div>
		<div class="Basic-Text-Frame">
		  <p class="Affiliations ParaOverride-1">PADESCA research Laboratory, Institute of Veterinary Sciences, University of Constantine 1, Algeria.</p>
		  <p class="Affiliations ParaOverride-1">&nbsp;</p>
		</div>
		<div>
		  <p class="Abstract ParaOverride-1"><span class="CharOverride-3">Abstract</span> | Honey is a natural product with numerous beneficial effects in human nutrition and medicine. However it may be contaminated with substances hazardous to the consumers such as antibiotics which are frequently used to treat bee colonies or to promote their growth. In this study we firstly optimized and validated the detection parameters of chloramphenicol and streptomycin in honey by High Performance Liquid Chromatography (HPLC), and then we conducted experiments to adopt a method of extraction of theses antibiotics residues from honey. The validation of our analytical method required the setting of multiples statistique tests. The coefficient of variation (CV) calculated for streptomycin and chloramphenicol is 1.89% and 3.85% respectively, indicating a good repetitiveness of our analytical methods. To evaluate reproducibility, we use Student Test at 5 % risk degree, the calculated “t<span class="CharOverride-4">”</span> is less than the “t<span class="CharOverride-4">”</span> table in the three sets of comparison (2.09). A good correlation between concentration and the response of the detector is attested by a correlation coefficient “r<span class="CharOverride-4">”</span> equal to 0.99 for chloramphenicol and 0.93 for streptomycin. The detection limit determined for streptomycin was 0.06 ppb and 0.024 ppm for chloramphenicol while limits of quantification are 0.2 ppb for Streptomycin and 0.08 ppm for Chloramphenicol. These results will be used later to identify and quantify these antibiotics residues in a large range of samples.</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
		  <p class="Abstract ParaOverride-1"><span class="CharOverride-3">Keywords</span> | Chloramphenicol, Streptomycin, Residues, Honey, HPLC</p>
		  <p>&nbsp;</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Editor----Citation"><span class="CharOverride-3">Editor</span> | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-3">Received</span> | February 22, 2015; <span class="CharOverride-3">Revised</span> | April 06, 2015; <span class="CharOverride-3">Accepted </span>| April 08, 2015; <span class="CharOverride-3">Published</span> | April 28, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-3">*Correspondence</span> | Bassem Chebira, Institute of Veterinary Sciences, University of Constantine 1, Algeria; <span class="CharOverride-3">Email: </span>Chebirab228@gmail.com</p>
			<p class="Editor----Citation"><span class="CharOverride-3">Citation </span>| Chebira B, Boultif L, Agabou A, Mekroud A (2015). Validation of a high performance liquid chromatography (HPLC) method to detect and quantify some antibiotics residues in honey. Adv. Anim. Vet. Sci. 3(5): 295-301.  </p>
			<p class="Editor----Citation"><span class="CharOverride-3">DOI </span>| <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.5.295.301"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.5.295.301</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-3">ISSN (Online)</span> | 2307-8316; <span class="Editor---Citation CharOverride-3">ISSN (Print)</span> | 2309-3331</p>
			<p class="Editor----Citation"><span class="CharOverride-3">Copyright</span> © 2015 Chebira 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>
			<p class="Editor----Citation">&nbsp;</p>
		</div>
<div class="Basic-Text-Frame">
		  <p class="Heading-1--Introduction----">INTRODUCTION </p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">A</span><span>ll </span>over the world and for thousands of years, humans in all societies had used bee products especially honey. This natural product contains about 200 phytochemicals and bioactive constituents such as antioxidants and enzymes involved in the defence mechanisms (<a href="#Babcan-S--Pivarnik-LF--Rand-AG--2002"><span class="Hyperlink">Babcan et al., 2002</span></a>; <a href="#Gheldof-N--Wang-XH--Engeseth-NJ--2002-"><span class="Hyperlink">Gheldof et al., 2002</span></a>; <a href="#Pontoh-J--Low-NH--2002-."><span class="Hyperlink">Pontoh et al., 2002</span></a>; <a href="#Bogdanov-S--Jurendic-T--Sieber-R--Gallmann-P.--2008-"><span class="Hyperlink">Bogdanov et al., 2008</span></a>; <a href="#Beretta-G--Gelmini-F--Lodi-V--Piazzalunga-A--Maffei-Facino-R--2010"><span class="Hyperlink">Beretta et al., 2010</span></a>; <a href="#Wang-J--Li-QX--2011-."><span class="Hyperlink">Wang and Li, 2011</span></a>). This composition leads to a large utilization of honey in folk and modern medicines as a remedy for treatment of wounds, allergies and gastrointestinal disorders (<a href="#Eteraf-Oskouei-T--Najafi-M--2013-."><span class="Hyperlink">Eteraf-Oskouei and Najafi, 2013</span></a>) because of its broad spectrum activity against some pathogenic and food-spoiling bacteria (<a href="#Al-Waili-NS--Haq-A--2004"><span class="Hyperlink">Al-Waili and Haq, 2004</span></a>; <a href="#Emsen-IM--2007-."><span class="Hyperlink">Emsen, 2007</span></a>) and its anti-neoplastic activity (<a href="#Sewllam-T--Miyanaga-N--Onozawa-M--Hattori-K--Kawari-K--Shimazui-T--Akaza-H--2003"><span class="Hyperlink">Sewllam et al., 2003</span></a>). </p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">However, use of honey in human nutrition and medicine could be hazardous, as this natural product may contain a variety of microorganisms such as <span class="CharOverride-4">Clostridium botulinum </span>(<a href="#Koluman-A--Meliko-lu-B--Derin-O--Okok-S--Anniballi-F--2013"><span class="Hyperlink">Koluman et al., 2013</span></a>), <span class="CharOverride-4">Bacillus cereus</span> (<a href="#L-pez-AC--Alippi-AM--2007-"><span class="Hyperlink">López and Alippi, 2007</span></a>), <span class="CharOverride-4">E. coli</span>, <span class="CharOverride-4">S. aureus</span> and <span class="CharOverride-4">Aspergillus sp </span>(<a href="#Dumen-E--Akkaya-H---z-GM--Sezgin-FH--2013"><span class="Hyperlink">Dumen et al., 2013</span></a>) and other contaminants, including heavy metals, radioactive materials, pesticides and antibiotics (mainly tetracyclines, streptomycin, sulfonamides, erythromycin, Lincomycin, Nitrofurans, Nitroimidazoles, Fluoroquinolones, Fumagillin, tylosin and chloramphenicol) which are widely applied to bee colonies, at relatively high doses as therapeutic agents, or at low sub-therapeutic doses as growth promoters (<a href="#Reybroeck-W--Daeseleire-E--De-Brabander-HF--Herman-L--2012-."><span class="Hyperlink">Reybroeck et al., 2012</span></a>). The presence of some antibiotics residues or their metabolites may result in toxic reactions in consumers while some others are able to generate hypersensitivity reactions (<a href="#Paige-LT--Tollefson-L--Miller-M--1997"><span class="Hyperlink">Paige et al., 1997</span></a>). Long term hazards (in relation with the relatively long half-life of antibiotic residues) include carcinogenicity, reproductive effects and teratogenicity at very low doses, which moreover increase the emergence of resistance in commensal and pathogenic bacteria leading to difficult-to-treat human infections (<a href="#Al-Waili-N--Salom-K--Al-Ghamdi-A--Ansari-MJ--2012"><span class="Hyperlink">Al-Waili et al., 2012</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">This is why honey has been subjected to many regulatory standards referred to as MRLs (Maximum Residue Limits). In the European Union and many other countries, no MRLs have been established for antibiotics in honey, resulting in a ‘zero tolerance’ for their residues implying that use of antibiotics is not allowed in apiculture and their residues should be absent in honey (<a href="#Michaud-V--2005-"><span class="Hyperlink">Michaud, 2005</span></a>; <a href="#Council-regulation-2377-90-EC-of-June-26th-1990"><span class="Hyperlink">Council regulation 2377/90/EC, 1990</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">Various analytical methods are used to identify and quantify these antibiotics residues in honey. Microbiological tests constitute easy techniques for their screening in routine control. Nevertheless, these tests have low detection capabilities and a high false-positive rate (<a href="#Gaudin-V--De-Courville-A--Hedou-C--Rault-A--Diomand--SE--Creff-Froger-C--Verdon-E.--2013"><span class="Hyperlink">Gaudin et al., 2013</span></a>). This leads to the development of other rapid qualitative or semi-quantitative tests such as Enzyme-Linking Immuno Sorbent Assays (ELISA) which give controversial results and require confirmation by more accurate, sensitive and reliable confirmatory methods such as gas or liquid chromatography combined or not to Mass Spectrometry (<a href="#Lopez-MI--Pettis-JS--Smith-IB--Chu-PS--2008-."><span class="Hyperlink">Lopez et al., 2008</span></a>; <a href="#Hammel-YA--Mohamed-R--Gremaud-E--Le-Breton-MH--Guy-PA--2008"><span class="Hyperlink">Hammel et al., 2008</span></a>). Recently a regenerable antigen microarray in combination with an automated flow injection system has been developed to identify and quantify antibiotic derivatives in honey (<a href="#Wutz-K--Niessner-R--Seidel-M--2011-."><span class="Hyperlink">Wutz et al., 2011</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">During 2011, the Algerian beekeeping industry counts 20 000 beekeepers and 1.5 millions of hives producing about 33000 quintals of honey. This local production is associated to an importation of about 150000 tons of honey from China, India and Saudi Arabia (<a href="#Oudjet-k--2012-"><span class="Hyperlink">Oudjet, 2012</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">In this paper, we aimed to optimize and validate an extraction method and various analysis parameters (stationary phase, mobile phase, absorption spectrum, flow rate and injected volume) to detect and quantify residues of Chloramphenicol and Streptomycin in honey commercialized in Constantine (North-east Algeria). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Heading-1--Introduction----">MATERIALS AND METHODS</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-">Instrumentation and Reagents</p>
		  <p class="Body-Text ParaOverride-1">The system is represented by an HPLC Shimadzu corporate device composed 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, pump 1 reference S/N:&nbsp;C20974009859J2, pump 2 references S/N:&nbsp;C21014009440CD), two UV detectors &nbsp;(models SPD-10Avp/10AVvp equipped with Deuterium lamp, absorption spectrum 190 to 350 nm, reference S/N:&nbsp;C21004001496LP), a gas cleaner:&nbsp;(Models DGU-20A5 reference S/N:&nbsp;L20244301983CR), a control system or integrator (models SCL-10 AVP, reference S/N:&nbsp;C21014009440CD), an injection loop, two reservoirs of mobile phase and a plastic piping &nbsp;allowing the progression of the liquid phase in the different compartments of the device. &nbsp;The other equipment consist of a centrifuge, a filtrating device, a cold chamber, analytical scale and HPLC special injection syringes (capacity 20 and 50 µL, reference C&nbsp;670-12554-03). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">All the chemicals used in this study were of ultra-residue analytical grade or of HPLC grade. Methanol (Ref. 5363C) and water (Ref. 34877) were obtained from Sigma-Aldrich and Acetonitrile (Ref. 6235A) from Biochem-Chemopharma.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text">Liquid chromatography solvents were ﬁltered with 0.45 µm Teﬂon and Nylon membranes before their use. Puriﬁed honey samples were successively ﬁltered through 0.45 µm X 47 mm Nylon ﬁlters (Supelco, Ref. Cat. 58061 &amp; 58062U).</p>
		  <p class="Body-Text">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">Standard of Sreptomycin (10ml. Ref. 85886) and Chlomphenicol (VETERANAL. 250mg. Ref. 46110) were purchased from Sigma-Aldrich.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">Sreptomycin standard stock solution was prepared by diluting 20 µg of the standard solution (1mg/ml) in 20 ml of methanol to get a concentration of 1ng/L.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">For chloramphenicol, 2 mg of the standard powder were melted in 10 ml of acetonitrile to obtain a stock solution with a concentration of 0.2 µg/L.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">To make the calibration curves, working solutions (different concentrations) were made by serially diluting the stock solutions in appropriate solvents every time just before use. Both standard and working solutions were stored at 4°C in amber flask until use.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">One hundred one (101), honey samples (125 g each) were collected at local markets in Constantine (North-East of Algeria) from September 2012 to August 2013. They were conveyed quickly in an isothermal tray to the laboratory and stored in darkness at 4°C until processing. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">Each honey sample was divided into 3 fractions of 10 g each. The first fraction served as a control; the second was doped with 0.2 mg of chloramphenicol while the latter was doped with 20 µg of streptomycin.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Heading-2--History-in-MM- ParaOverride-1">Optimization and Validation of Chromatographic Analysis </p>
		  <p class="Body-Text ParaOverride-1">In order to identify and quantify streptomycin and chloramphenicol residues in honey by HPLC, we started at first with an optimization of the analysis conditions [stationary phase, mobile phase, absorption spectrum (wavelengths), flow rate and injection volumes]. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">The accuracy of the method is verified by statistical calculations of repeatability and reproducibility (according to the <a href="#CEAEQ--Centre-d-Expertise-en-Analyse-Environnementale-du-Qu-bec---2007-"><span class="Hyperlink">CEAEQ, 2007</span></a>). For repeatability, calculation of the coefficient of variation is a very good indicator of the analytical method precision, while for reproducibility we carried out in addition to the coefficient of variation, the comparison of averages using Student’s “<span class="CharOverride-4">t</span><span class="CharOverride-7">”</span> test at P&lt;5%</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">To test the repetitiveness of our method, we performed a series of 10 essays on the same day, with the same work solution, the same operator, the same place, the same equipment and under the same analysis conditions (According to ISO 3534-1 and ISO 5725-1 standards).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text">To achieve the test of reproducibility, two different operators carried out three series of twenty essays each, with three different work solutions (one for each series) on different days (according to ISO 3534-1 and ISO 5725-1 standards).</p>
		  <p class="Body-Text">&nbsp;</p>
		  <p class="Body-Text">For verifying the linearity of our analytical method, we have prepared a range of calibrations for each antibiotic. They consist of five standards of chloramphenicol containing respectively 12%, 25%, 50%, 75% and 100% of the concentration determined during the optimization step. For streptomycin, the calibration range has four standards corresponding to 25%, 50%, 75% and 100% of the optimization (according to ISO 3534-1 and ISO 5725-1 standards).</p>
		  <p class="Body-Text">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">To determinate the minimum of detection limit (MDL) we used a concentration equivalent to 3 times the standard deviation at low level of each antibiotic standard in its proper solvent (MDL = 3 × S. S: standard deviation) (according to ISO 3534-1 and ISO 5725-1 standards).</p>
			<p class="Body-Text ParaOverride-1">The method quantification limit (MQL) was evaluated by use a concentration  equal to 10 times the standard deviation obtained during MDL determination (MQL = 10 × S. S: standard deviation) (according to ISO 3534-1 and ISO 5725-1 standards).</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 from honey matrix, the preparation of the sample is a crucial step. The adopted extraction method of streptomycin and chloramphenicol from honey is based on the procedure described by <a href="#Cai-Y--Cheng-J--Mou-S--Yiqiang-L--2005-"><span class="Hyperlink">Cai et al. (2005)</span></a> and it consists of the following steps: 10 g of honey were mixed with 20 ml of acetonitrile and 10 ml of LC grade water and vortexed for 1 min. &#160;The mixture is then centrifuged at 6000 rpm for 10 min and the supernatant is collected and passed through Whatman filters. This filtrate supplemented with 10 ml of acetonitrile undergoes a vortex-homogenization for 2 minutes then a centrifugation at 3000 rpm for 5 mn. Finally, the supernatant is harvested and filtered firstly with a Whatman filter, then with a Nylon membrane filter (Supelco: 0.45 µm X 47 mm).&#160;This procedure was applied on all doped portions of honey samples. </p>
			<p class="Body-Text ParaOverride-1">To assess the efficacy of this procedure in the extraction of Streptomycin and Chloramphenicol residues from the honey matrix, 20 series of trials were conducted on honey samples doped with each antibiotic standard as recommended by <a href="#Diaz-TG--Cabanillas-AG--1990"><span class="Hyperlink">Diaz and Cabanillas (1990)</span></a>, <a href="#Helio-A--Martins-J--Tereza-A--Kussumi-A--Wang-D--Lebre-T--2007-."><span class="Hyperlink">Helio et al., (2007)</span></a> and <a href="#Vinas-P--Balsalobre-N--Hernandez-Cordoba-M--2007"><span class="Hyperlink">Vinas et al. (2007</span><span class="Hyperlink CharOverride-9">)</span></a><span class="CharOverride-9">.</span></p>
			<p class="Heading-2--History-in-MM-">Statistics Study</p>
			<p class="Body-Text ParaOverride-1">We realized an average comparison according to Student ‘‘<span class="CharOverride-4">t</span>’’  Test. &#160;We calculated the average, the standard deviation and the variance from the obtained surfaces to calculate “<span class="CharOverride-4">t</span>” (calculated) and to compare it with the “<span class="CharOverride-4">t</span>” (table of Student) at the respective risks of d 5%. &#160;</p>
			<p class="Body-Text ParaOverride-1">The “<span class="CharOverride-4">t</span>” (calculated) is measured between every two series (<span class="CharOverride-4">a</span> and <span class="CharOverride-4">b</span>, <span class="CharOverride-4">a</span> and <span class="CharOverride-4">c</span>, <span class="CharOverride-4">b</span> and <span class="CharOverride-4">c</span>) according to the following formula: </p>
			<p class="Body-Text ParaOverride-2"><span class="CharOverride-4">t = |Xa - Xb|/√ </span><span class="CharOverride-10">σ</span><span class="CharOverride-4">²/Na + </span><span class="CharOverride-10">σ</span><span class="CharOverride-4">²/Nb</span></p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-4">Xa</span> being the average of the first series (series <span class="CharOverride-4">a</span>) - <span class="CharOverride-4">Xb</span> being the average of the second series (series <span class="CharOverride-4">b</span>) - <span class="CharOverride-10">σ</span><span class="CharOverride-4">²</span> being the variance between the two series <span class="CharOverride-4">a</span> and<span class="CharOverride-4">b</span> - <span class="CharOverride-4">Na</span> being the number of the repetitions of the series <span class="CharOverride-4">a</span>&#160;- <span class="CharOverride-4">Nb</span> being the number of the repetitions of the series <span class="CharOverride-4">b</span>.&#160;</p>
			<p class="Body-Text ParaOverride-1">If “t<span class="CharOverride-4">”</span> is inferior to “<span class="CharOverride-4">t</span>”  table, the difference between the two series is not significant which means that our method is repeatable or reproducible.&#160;</p>
			<p class="Body-Text ParaOverride-1">If the calculated “<span class="CharOverride-4">t</span>” is superior to “<span class="CharOverride-4">t</span>” table then the difference between the two series is significant and our method is not reliable.&#160;</p>
			<p class="Body-Text ParaOverride-1">All tests of the statistic study were made using the software Stagraphics version 15.2.0.6&#160;(2010).</p>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-12">RESULTS AND DISCUSSION</span></p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Withheld Chromatographic Analysis Conditions</p>
			<p class="Body-Text ParaOverride-1">Our experimental investigations allowed keeping the following HPLC analysis parameters:</p>
			<ul>
				<li class="Body-Text ParaOverride-3">For both antibiotics, all analyses were performed in isocratic mode with a reverse-phase C18 Vp - ODS column (Supelco, Supelcosil: 250 mm x 4.6 mm 5 µmo). </li>
				<li class="Body-Text ParaOverride-3">For chloramphenicol, the mobile phase consisting of 20 % methanol and 80 % LC grade water was pumped at a flow rate of 1.2 ml/min. Detection was performed at 272 nm (UV detector). The injected volume was 10 µl and chromatography was performed at ambient temperature during 10 min.</li>
				<li class="Body-Text ParaOverride-3">For streptomycin, the following analytical conditions are retained: mobile phase entirely composed of methanol, Injected volume: 10 µl, wavelength (UV detector): 280 nm, flow rate: &#160;1.2 ml/min and analysis time: 10 min at ambient temperature. &#160;</li>
			</ul>
			<p class="Heading-2--History-in-MM-">Examination of the Method’s Reliability</p>
			<p class="Body-Text ParaOverride-1">This was conducted to verify the efficacy of the withheld parameters.</p>
			<p class="Heading-3">Repeatability</p>
			<p class="Body-Text ParaOverride-1">The coefficients of variation (CV) calculated for streptomycin and chloramphenicol are 1.89% and 3.85% respectively (<span class="Hyperlink">Table 1</span>), indicating a good repetitiveness of our analytical methods. </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
	<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-3"><a id="Table-1--159"></a>Table 1:</span> Statistical results of repeatability for streptomycin (Str) and chloramphenicol (Cap)</p>
			<table width="657" 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-1">
						<td />
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Number </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-14">of trials</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Average</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Standard </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-14">deviation</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Coefficient of</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-14">variation (CV)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Str</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">663289.7</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">12563.78</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">1.89%</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Cap</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3782646.4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">145733.37</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.85%</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-3"><a id="Table-3-"></a></span><span class="CharOverride-17">T</span><span class="CharOverride-17">hese findings are in agreement with other works in which coefficients of variation were less than 10% (Vinas et al., 2007). Benetti et al. (2004) found a coefficient of variation equal to 7.5% for three series of tests carried out on the same day and Ortelli et al. (2004) while working on honey samples spiked with a standard chloramphenicol at a concentration of 5 µg/kg reported a CV of 5.3% for a series of 4</span><span class="CharOverride-17"> trials.</span></p>
			<p class="Heading-3">&nbsp;</p>
			<p class="Heading-3">Reproducibility</p>
			<p class="Body-Text ParaOverride-1">The recorded coefficients of variation are between 3.10 and 3.46% for the chloramphenicol and between 6.41 and 10.53% for streptomycin (<a href="#Figure-2-"><span class="Hyperlink">Table 2</span></a>). </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">These values are consistent with the recommendations of the manual of the <span class="CharOverride-4">Codex Alimentarius Commission</span>, which sets at 35% the maximum value of the calculated coefficient of variation for the inter-assay so that the analysis method could be considered to be reliable. These results are significantly lower than the coefficient of variation granted by <a href="#Commission-decision-2002-657-EC"><span class="Hyperlink">decision 2002/657/EC</span></a> of the European Union for the validation of the analytical methods on animal foodstuffs, which is set at 12%.</p>
			<p class="Body-Text ParaOverride-1">As mentioned in <span class="Hyperlink">Table 3</span>, the calculated “t<span class="CharOverride-4">”</span> is less than the “t<span class="CharOverride-4">”</span> table in the three sets of comparison, confirming the method’s reproducible in our analysis conditions.</p>
			<p class="Heading-3">&nbsp;</p>
			<p class="Heading-3">Linearity</p>
			<p class="Body-Text ParaOverride-1">A good correlation between concentration and the response of the detector is attested by a correlation coefficient “r<span class="CharOverride-4">”</span> equal to 0.99 for chloramphenicol and 0.93 for streptomycin (<a href="#Figure-1-"><span class="Hyperlink">Figures 1</span></a> and <a href="#Figure-2-"><span class="Hyperlink">2</span></a>) which is a good indicator of linearity according to European Council regulation 2377/90/EC. The respective regression equations for the standard chloramphenicol and streptomycin are: </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-2"><span class="CharOverride-4">Y = 3.77405e-8 X + b / b = 0</span> </p>
			<p class="Body-Text ParaOverride-1">and </p>
			<p class="Body-Text ParaOverride-2"><span class="CharOverride-4">Y = 3.12846e-6 X + b / b = 0</span>.</p>
			<p class="Body-Text ParaOverride-2">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Limits of Detection and Quantification of the Method</p>
			<p class="Body-Text ParaOverride-1">The detection limit determined by the points covered by the peak area is 37691.34 points (0.06 ppb) for streptomycin and 437199.33 points (0.024 ppm) for chloramphenicol while limits of quantification are about 125&nbsp;637.8 points (0.2 ppb) for Streptomycin and 1457333.7 points (0.08 ppm) for Chloramphenicol (<a href="#Table-4-"><span class="Hyperlink">Table 4</span></a>).</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150501144352.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150501144352.png" width="80" height="80"></a>
            
            <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-3"><a id="Figure-1-"></a>Figure 1: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150501144352.png"> Chloramphenicol calibration curve. Standard concentrations from 0.0125 to 0.2mg/ml</a></p>
       </div>

			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-3"><a id="Table-2-"></a>Table 2: </span>Statistical results of reproducibility for streptomycin (Str) and chloramphenicol (Cap)</p>
			<table width="657" class="Table-Style-1" id="table-2">
            
				<colgroup>
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td rowspan="2" />
						<td colspan="2">
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-14">           Series  1</span></p>
						</td>
						<td colspan="2">
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-14">      Series  2</span></p>
						</td>
						<td colspan="2">
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-14">        Series  3</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Str</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Cap</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Str</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Cap</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Str</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Cap</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Number of trials</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">20</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">20</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">20</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">20</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">20</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">20</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Average</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">101505.7</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.94196E6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">96885.8</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.90143E6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">93465.6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.86921E6</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Standard deviation</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">6903.51</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">122303.0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10211.4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">135123.0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">5999.18</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">124151</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Coefficient of variation (CV)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">6.80</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10.53</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.46</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">6.41</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.20</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Minimum</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">91787.0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.69529E6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">78388.0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.46</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">84920.0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3.63</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Maximum</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">117000.1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">4.1486E6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">113552.6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">4.14</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">107786.9</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">4.11</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-3"><a id="Table-3--159"></a><a id="Table-3--160"></a>Table 3: </span>Comparisons of averages and “<span class="CharOverride-4">t</span>’’ tests for chloramphenicol (Cap) and streptomycin (Str)</p>
			<table  width="657" id="table-3" class="Table-Style-1">
				<colgroup>
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td />
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Averages </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Comparison</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Calculated “t’’ test (ct)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Table “t’’ test (tt)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Risk </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-14">degree</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">ct vs tt</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td rowspan="3">
							<p class="Basic-Paragraph"><span class="CharOverride-16">Cap</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">a/b</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0,994757</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">2.09</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">5%</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">tt&gt;tc</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">b/c</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0,78513</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">2.09</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">5%</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">tt&gt;tc</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">a/c</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0,78513</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">2.09</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">5%</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">tt&gt;tc</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td rowspan="3">
							<p class="Basic-Paragraph"><span class="CharOverride-16">Str</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">a/b</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">1,67602</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">2.09</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">5%</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">tt&gt;tc</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">b/c</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">1,29151</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">2.09</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">5%</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">tt&gt;tc</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">a/c</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">1,93115</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">2.09</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">5%</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">tt&gt;tc</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-3"><a id="Table-4-"></a>Table 4: </span>Minimum detection and quantification limits</p>
			<table id="table-4" class="Table-Style-1">
				<colgroup>
					<col />
					<col />
					<col />
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td rowspan="2" />
						<td colspan="2">
							<p class="Basic-Paragraph ParaOverride-2"><span class="CharOverride-14">Streptomycin</span></p>
						</td>
						<td colspan="2">
							<p class="Basic-Paragraph ParaOverride-2"><span class="CharOverride-14">Chloramphenicol</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Value in point area</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Value in ng/kg</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Value in point area</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Value in µg/kg</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">No. of tests</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Average (m)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">663289.7</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3782646.4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0.2</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Standard deviation (S)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">12563.78</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0.02</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">145733.37</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0.008</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Minimum detection limit </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-16">MDL=S x 3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">37691.34</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0.06</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">437199.33</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0.024</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Minimum quantification limit </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-16">MQL=S x 10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">125&#160;637.8</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0.2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">1457333.7</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">0.08</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150501141153.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150501141153.png" width="80" height="80"></a>
            
           <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-22"><a id="Figure-2-"></a>Figure 2: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150501141153.png">Streptomycin calibration curve. Standard concentrations from 0.08 to 0.33µg/ml</a></p>
       </div>
			
			<p class="Heading-2--History-in-MM-">Extraction Method</p>
			<p class="Body-Text ParaOverride-1">After doping honey samples with the streptomycin and chloramphenicol standards, the adopted extraction procedure allows obtaining a transparent and homogenous liquid which is injected directly in the HPLC device. We obtained corresponding peaks after 2.55 min for chloramphenicol (<a href="#Figure-3-"><span class="Hyperlink">Figure 3</span></a>) and 2.627 for streptomycin (<a href="#Figure-4-"><span class="Hyperlink">Figure 4</span></a>) while for the negative control sample, no peak can be identified (<a href="#Figure-5-"><span class="Hyperlink">Figure 5</span></a>).</p>
			<p class="Body-Text">Appearance times of the obtained peaks correspond ex-</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150501143953.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150501143953.png" width="80" height="80"></a>
            
           <p class="Body-Text ParaOverride-1"><span class="CharOverride-22"><a id="Figure-3-"></a>Figure 3: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150501143953.png">Chromatogram of honey doped with chloramphenicol standar</a></p>
       </div>
			<br>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150501140454.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150501140454.png" width="80" height="80"></a>
            
           <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-3"><a id="Figure-4-"></a>Figure 4: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150501140454.png">Chromatogram of honey doped with streptomycin standard</a></p>
       </div>
       <br>
			
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150501142554.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150501142554.png" width="80" height="80"></a>
            
           <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-3"><a id="Figure-5-"></a>Figure 5: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150501142554.png">Chromatogram of negative control honey samples</a></p>
       </div>
       
			<br>
			<p class="Body-Text ParaOverride-1">actly to the retention times obtained from the tests on the internal standard. This indicates that chloramphenicol and streptomycin extracted from doped samples and analysed with the same optimized parameters had been recognized by our HPLC system and confirms the accurateness of the adopted extraction procedure. </p>
			<p class="Body-Text ParaOverride-1">Different methods of extraction have been described in several previous studies (<a href="#Diaz-TG--Cabanillas-AG--1990"><span class="Hyperlink">Diaz and Cabanillas, 1990</span></a>; <a href="#Cai-Y--Cheng-J--Mou-S--Yiqiang-L--2005-"><span class="Hyperlink">Cai et al., 2005</span></a>; <a href="#Helio-A--Martins-J--Tereza-A--Kussumi-A--Wang-D--Lebre-T--2007-."><span class="Hyperlink">Helio et al., 2007</span></a>; <a href="#Vinas-P--Balsalobre-N--Hernandez-Cordoba-M--2007"><span class="Hyperlink">Vinas et al., 2007</span></a><span class="CharOverride-9">).</span></p>
			<p class="Heading-1--Introduction----">CONCLUSION</p>
			<p class="Body-Text ParaOverride-1">The preliminary results are very satisfactory. Our HPLC system is able to detect the residues of streptomycin and shloramphenicol in spiked honey samples after an efficient extraction procedure.</p>
			<p class="Body-Text ParaOverride-1">Tests carried out for the validation of our method of chromatographic analysis revealed very reliable and accurate results confirmed by good repetitiveness, good precision and linearity as well as a valuable coefficient of variation.</p>
			<p class="Body-Text ParaOverride-1">These encouraging results will serve as a basis for further experimental investigations on contamination with antibiotics in a large range of locally produced or imported honey samples.</p>
			<p class="Heading-1--Introduction----">Acknowledgment</p>
			<p class="Body-Text ParaOverride-1">This work is dedicated to the memory of our laboratory director Pr. Elhadef-Elokki Saadoune. A beloved father, friend, colleague, teacher and researcher who left us forever and far too soon.</p>
			<p class="Heading-1--Introduction----">Conflict of Interest </p>
			<p class="Body-Text ParaOverride-1">The authors declare that they have no conflict of interest.</p>
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