International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 4(5), 59-65, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 59 Quantitative Analysis of Oxytetracycline Residues in Honey by High Performance Liquid ChromatographyChilumuru Rama Mohana Rao1*, Lakkineni Cyril Arun Kumar and Chandra Bala SekharanDepartment of Biotechnology, VSR and NVR College, Tenali, Andhra Pradesh, INDIA Department of Zoology, VSR and NVR College, Tenali, Andhra Pradesh, INDIA Department of Pharmaceutical Biotechnology, Medarametla Anjamma Mastan Rao College of Pharmacy, Narasaraopet, INDIA Available online at: www.isca.in, www.isca.me Received 19th March 2015, revised 20th April 2015, accepted 8th May 2015 Abstract In this study, a simple, sensitive, selective and precise HPLC method is developed for the determination of oxytetracycline in bulk and honey samples. Separation of oxytetracycline was achieved on a Kromosil C18 analytical column (250 mm × 4.6 mm I.D., 5 m particle size). The mobile phase was a mixture of acetonitrile and water (85:15 v/v) at a flow rate of 1.5 ml/min. The method was linear in the range of 1–6 g/ml. The LOD and LOQ values are found to be 0.05 and 0.10 g/ml, respectively. The developed method was successfully applied for the determination of the oxytetracycline in honey sample following extraction of the oxytetracycline with McIlvaine buffer and Solid Phase Extraction system. All the honey samples that were analyzed for oxytetracyline residues had higher residue levels than the recommended maximum residue level for honey. The method described in this study would be useful for routine monitoring of oxytetracycline residues in honey. Keywords: Oxytetracycline, HPLC, honey, european union regulations.Introduction Antibiotics are among the most frequently prescribed medications in modern medicine. Antibiotics cure disease by killing or injuring bacteria. Today, over 100 different antibiotics are available to cure minor as well as life-threatening infections. Broad spectrum antibiotics are effective against a broad range of microorganisms in comparison to narrow spectrum antibiotics. In the livestock industry, antibiotics are used abundantly. There are three main uses of antibiotics in livestock production. Antibiotics can be used as a therapeutic, as a growth promoter and as a prophylactic. Therefore antibiotics are necessary to society-used properly or not. The commercial importance of antibiotics is not only for treating infections in humans but also for food production and keeping animals and plants disease free. Antibiotics are screened for any negative effects on humans or other mammals before approval for clinical use. However, some antibiotics have been associated with a range of adverse effects. Side effects range from mild to very serious depending on the antibiotics used, the microbial organisms targeted and the individual patient. Adverse effects range from fever and nausea to major allergic reactions including photodermatitis and anaphylaxis. Honey is a mixture of sugars and other compounds. With respect to carbohydrates, honey consists of mainly fructose (about 38.5%) and glucose (about 31%), making it similar to the synthetically produced inverted sugar syrup, which is approximately 48% fructose, 47% glucose, and 5% sucrose. It contains only trace amounts of vitamins or minerals. Honey also contains minute amounts of compounds thought to function as antioxidants like chrysin, pinobanksin, vitamin C, catalase, and pinocembrin. The specific composition of any batch of honey depends on the flowers available to the bees that produced the honey. Although the mechanism of its action is not fully understood, since ancient times honey has been widely used throughout the world as a healing medicine7,8. It is mainly used for the treatment of eye diseases, cough, thirst, phlegm, hiccups, and blood vomit, leprosy, diabetes, obesity, worm infestation, vomiting, asthma, diarrhoea and healing wounds and also used as a natural preservative and sweetener in many ayurvedic preparations. Due to less production of honey, northern countries importing honey from eastern countries. Antibiotics have been added to honey as preservatives by the honey exporters9,10. Oxytetracycline (figure-1) is a broad-spectrum antibiotic commonly used in human and veterinary medicine for therapeutic and prophylactic purposes. In apiculture beekeepers use oxytetracycline against the bacterial diseases that affects honey bees11. As a result, oxytetracycline residues can be detected at trace levels in honey of treated bees. Oxytetracycline residues show a relatively long half-life and may show direct toxic effects on consumers11. Therefore, the presence and maximum residue values of oxytetracycline residues in honey should be regulated. Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 59-65, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 60 OHOHCHOHOHCHOH Figure-1 Chemical structure of oxytetracycline Different analytical methods are reported for the determination oxytetracycline residues in honey. These include HPLC with UV detection12-17, HPLC with fluorescence detection18-22, Liquid chromatography with mass spectrometry11,23-26, capillary high performance liquid chromatography27, HPTLC28, high performance capillary electrophoresis29, Immunological assay30, Capillary zone electrophoresis31 and spectrophotometric methods32. The above reported methods suffers from one or more drawbacks such as use of internal standard, expensive detectors, cumbersome procedure, long analysis time, less sensitive, lack of precision and accuracy. Hence, an attempt has been made to develop a simple, sensitive, cost effective and reliable HPLC with UV detection method for estimation of oxytetracycline residues in honey without the use of internal standard. Material and Methods Mobile phase: The chemicals and solvents used for the preparation of mobile phase are of HPLC grade. Milli-Q-water was used throughout. The mobile phase used for the analysis of oxytetracycline consists of a mixture of acetonitrile and water in the ratio of 85:15 v/v. The mobile phase is also used as diluent for the preparation of oxytetracycline standard solutions. The mobile phase was filtered through a 0.45 m Millipore membrane filter and sonicated for 15 minutes for degassing prior to use. Instrumentation and chromatographic conditions: Chromatographic separation was performed on an isocratic High Pressure Liquid Chromatography system (Shimadzu HPLC class VP series, Shimadzu Corporation, Kyoto, Japan) with two LC-10 AT, VP pumps, variable wavelength programmable UV/Visible detector SPD-10A, VP, CTO-10AS VP column oven, SCL-10A, VP system controller. A 20 l Hamilton syringe was used for injecting the samples. Data were analyzed by using PEAK software. Double beam UV-VIS spectrophotometer Model UV-VIS 2301 (Tech-comp limited, Hong Kong, Japan) was used for spectral studies. Degassing of the mobile phase was done by using Ultrasonic Bath Sonicator (Loba Chemie Pvt. Ltd. Mumbai, India). Samples were weighed by using Denver electronic Weighing Balance (Denver instruments, Colorado, USA.). Separation was achieved isocratically with a Kromosil C18 analytical column (250 mm × 4.6 mm I.D., 5 m particle size), under reversed phase chromatographic conditions, eluted with a mixture of acetonitrile and water (85:15 v/v) as the mobile phase at flow rate of 1.5 ml/min. Detection was carried out by absorbance at 360 nm. The analysis was carried out at an ambient temperature and injection volume was 20 l. Standard solutions: The bulk form of oxytetracycline was obtained from Matrix Laboratories Limited, Hyderabad, India and was used as received. The stock standard solution of oxytetracycline (1 mg/ml) was prepared in mobile phase. Six series of oxytetracycline working standard solutions at the concentration values of 1, 2, 3, 4, 5 and 6 g/ml were prepared from the stock standard solution by apt dilution with the mobile phase. Sample collection: Seven different brands of honey samples were collected from local markets of Tenali, Guntur Dt. Andhra Pradesh, India. For each brand 6 honey samples were collected. Raw honey sample was collected from Repalle, Guntur, Dt,Andhra Pradesh, India. The samples were stored at 20°C until analysis. Extraction of oxytetracycline residues from honey samples: The oxytetracycline residues from honey samples were extracted with optimized extraction method as described by Pagliuca G. et. al.33. Five gram honey sample was taken and dissolved in 20 ml of 0.1 M NaEDTA-Mcllvaine buffer at pH 4. The solution was vortexed for 5 minutes, filtered and made ready for Solid Phase Extraction (SPE) clean-up procedure. After extraction, 60 mg of sample was loaded on a Sampli Q OPT 3 ml cartridge previously conditioned with 1 ml methanol and 1 ml water. The SPE cartridge was then washed with 10 ml water. Finally, the sample was eluted with 1 ml ethyl acetate (Sdfine-Chem limited, Mumbai, India) directly in sample tube. After evaporating the solvent at 40şC under nitrogen stream, the residues were reconstituted with 1 ml of mobile phases. General analytical procedure: Working standard solutions equivalent to 1 to 6 g/ml of oxytetracycline were prepared by appropriate dilution of the stock standard solution with the mobile phase. Twenty l aliquot of each solution was injected into the column in triplicate. The mobile phase was pumped from the solvent reservoir to the column at a flow rate of 1.5 ml/min. The peaks were recorded at 360 nm. The linearity curve was constructed by plotting peak area versus concentration of the oxytetracycline. The concentration of the unknown was read from the calibration graph or computed from the regression equation derived using the mean peak area-concentration data Quantification of oxytetracycline in honey samples: The extract prepared from honey samples, as described in section “Extraction of oxytetracycline residues from honey samples”, Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 59-65, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 61 was further diluted appropriately with the mobile phase for the analyses of oxytetracycline residues by the proposed method. The honey sample solution was injected into HPLC system in duplicate. The chromatograms were recorded. The area under the peak was calculated. The concentration of oxytetracycline in the honey samples were calculated using the corresponding calibration curve or corresponding regression equation. Results and Discussion Method Development: A series of trials was conducted, with different analytical columns and with varied proportions of the organic solvent and water, to develop an appropriate reverse phase liquid chromatographic method for quantification of oxytetracycline in bulk and honey samples. Finally a typical chromatogram with better peak shape and low retention time was obtained with acetonitrile and water in the ratio of 85:15 v/vat a flow rate of 1.5 ml/min. The chromatographic separation was performed on Kromosil C18 analytical column (250 mm × 4.6 mm I.D., 5 m particle size) by injecting 20 l and analyte was detected with ultraviolet detector set at 360 nm. The retention time of oxytetracycline was found to be 6.81 minutes. The optimized conditions were given in table-1. Table -1 Optimized chromatographic conditions Parameter Optimized condition Column Kromosil C18 (250 mm × 4.6 mm I.D., 5 m particle size) Mobile phase Acetonitrile: Water (85:15 v/v) Flow rate 1.5 ml/min Detection wavelength 360 nm Injection volume 20 l Column temperature 25 ± 1C Retention time 6.81 minutes Method validation: The validation was performed with above developed RP-HPLC method for estimation of oxytetracycline in bulk and honey samples according to ICH guidelines. Various parameters were evaluated such as system suitability, linearity, sensitivity, selectivity, precision and accuracy. System Suitability: System suitability was performed to prove the adequacy of the resolution and repeatability of the system. System suitability was assessed by injecting six replicate injections of the oxytetracycline working standard solution (6 g/ml) and parameters such as peak area, theoretical plates, retention time, and peak asymmetry were evaluated. The percentage relative standard deviation for the parameters was determined. The results were shown in table-2. The results reported are within the limits. Table-2 System suitability parameters Parameter Mean value% RSDAcceptable limits Retention time 6.81 1.158 % RSD 2.0 Peak area 6701 0.269 % RSD 2.0 Theoretical plates (N) 4513 0.429 N � 2000 Peak asymmetry (P) 1.45 0.592 P 1.5 * Average of five determinations Linearity: To assess the linearity of the proposed method, calibration curve were constructed by plotting the peak area of the working standard solutions in the range of 1-6 g/ml vs the oxytetracycline concentration. The regression data of six series of calibration curve are indicated in table- 3. From the results obtained the proposed method was found to be linear. Table-3 Linearity and regression data ParameterValue Linearity range g/ml) 1-6 Regression equation (Y = a + bc): Slope (b) 1112.85 Intercept (a) 65.857 Correlation Coefficient (r) 0.9996 Sensitivity: The sensitivity of the method was assessed by determining the parameters like limit of detection (LOD) and limit of quantification (LOQ). The LOD and LOQ for the oxytetracycline were based on the lowest detectable peak that had signal/noise = 3 and 10, respectively. The obtained values (LOD = 0.05 g/ml and LOQ = 0.10 g/ml) were satisfactory and indicate that the method is sensitive. Selectivity: The selectivity of the method was established by checking the potential interference with the components from the mobile phase and honey sample. The chromatograms of pure oxytetracycline, blank mobile phase and honey sample are recorded and compared (figure-2, 3 and 4). No interference was observed for any of the components from the mobile phase and honey sample. The results indicated the selectivity of the method for the assay of oxytetracycline in honey sample. Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 59-65, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 62 Figure-2 Chromatogram of pure oxytetracycline Figure -3 Chromatogram of oxytetracycline in honey Figure-4 Chromatogram of mobile phase blank Precision: The method precision studies were carried out by injecting six replicates of oxytetracycline working standard Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 59-65, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 63 solutions with the same concentration (6 g/ml). The percentage relative deviation of the peak areas was calculated from the chromatograms and results (RSD = 0.596%) obtained were within the limits of 2%. Therefore, the proposed method was found to be precise. Accuracy: The accuracy of the method was assessed by calculating the recovery studies of the oxytetracycline at three different concentration levels (50%, 100%, and 150%) by standard addition method. A known amount of oxytetracycline (within linearity range) was added to prequantified sample solution. Three replicates of each concentration were injected into the HPLC system. The mean percentage recovery of oxytetracycline was varied between 99.00 and 99.80%. The recovery results indicating that the developed method was found to be accurate. The results were shown in table- 4.Table- 4 Percentage recovery results of oxytetracycline Level (%) Concentration of oxytetracycline (g/ml) Recovery (%) Average recovery (%) Fixed Spiked Recovered 50 2 1 2.97 99.00 99.43 100 2 2 3.98 99.50 150 2 3 4.99 99.80 Application of the method for the determination of oxytetracycline in honey samples: The developed and validated method was successfully applied for the quantification of oxytetracycline in honey samples. The results were summarized in table-5. For the present study, 7 branded honey samples and one raw honey sample was collected from the local markets at Tenali and Repalli, respectively. Table -5 Assay of oxytetracycline in honey samplesS.No Sample Branded honey* Concentration of oxytetracycline (µg/kg) 1 Sample -1 Brand - 1 0.12 ± 0.25 2 Sample -2 Brand - 2 0.09 ± 0.43 3 Sample -3 Brand - 3 0.17 ± 0.57 4 Sample -4 Brand - 4 0.08 ± 0.96 5 Sample -5 Brand - 5 0.17 ± 0.57 6 Sample -6 Brand - 6 0.11 ± 0.39 7 Sample -7 Brand - 7 0.05 ± 0.48 8 Sample -8 Raw honey ** 0.54 ± 0.49 * All the branded honey samples were collected at local markets of Tenali ** Raw honey was collected from Repalli Oxytetracycline was detected in all the honey samples. The concentration of oxytetracycline was found to be high in raw honey sample (0.54 g/kg) followed by brand 5 and 3 (0.17 g/kg), brand 1 (0.12 g/kg), brand 6 (0.11 g/kg), brand 2 (0.09 g/kg), brand 4 (0.08 g/kg) and brand 7 (0.05 g/kg) samples. The European Union Regulations 396/2005 (2013) has recommended zero level tolerance for oxytetracycline residues in honey. However, the oxytetracycline residues were higher than maximum residue limit established by European Union Regulations34. Conclusion A rapid, simple, accurate, precise, and selective HPLC with UV detection method has been developed for the quantification of oxytetracycline and honey samples. The developed method was successfully employed for the simple and rapid determination of oxytetracycline in honey samples with good precision and accuracy. The results of assay of the oxytetracycline in the selected honey samples indicated that in most of the collected samples the antibiotic residues were higher than the maximum residue limits set by European Union Regulations. Thus the proposed method is useful in the monitoring and quantification of the oxytetracycline in honey samples. References 1.Ahmet N. and Emel N., Phagotrophic protozoa, A new weapon against pathogens, Med. Hypo., 70(1), 141-142 (2008)2.Kirkup B.C. Jr., Bacteriocins as oral and gastrointestinal antibiotics, theoretical considerations, applied research, and practical applications, Current Med. Chem.,13(27), 3335-3350 (2006)3.Slama T.G., Amin A., Brunton S.A., File T.M., Milkovich G., Rodvold K.A., Sahm D.F., Varon J. and Weiland D., Council for appropriate and rational antibiotic therapy (CARAT), a clinician's guide to the appropriate and accurate use of antibiotics: the council for appropriate and rational antibiotic therapy (CARAT) criteria, American J. Med.,118(Suppl, 7A), 1S-6S (2005)4.James D.T. and Carol M.A., The law of unintended consequences and antibiotics, Open J. Immuno.,2(2), 59-64 (2012)5.Gheldof N., Wang X. and Engeseth N., Identification and quantification of antioxidant components of honeys from various floral sources, J. Agri. Food Chem., 50(21), 5870–5877 (2002)6.Martos I., Ferreres F. and Tomás-Barberán F., Identification of flavonoid markers for the botanical Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 59-65, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 64 origin of Eucalyptus honey, J. Agri. Food Chem.,48(5), 1498–502 (2000)7.Carla B., The detection of C4 sugars in honey, Canadian Honey Council, 12(1), 37-40 (1999) 8.Gabriel V.L., Charaka samhitam, Hand book on Ayurveda, Volume I, 20039.Kaufmann A., Pacciarelli B., Prijic A., Ryser B. and Roth S., Travaux de chimie alimentaire et d’hygične, 90 , 167-176 (1999)10.Diserens J.M. and Savoy M.C., Nestlé research center, personal communication, (2000)11.Gunes M.E., Gunes N. and Cibik R., Oxytetracycline and sulphonamide residues analysis of honey samples from southern marmara region in turkey, Bulgarian J. Agri. Sci.,15(2), 163-167 (2009)12.Galeano D.T., Guiberteau C.A. and Salinas F., Rapid determination of sulfathiazole, oxytetracycline and tetracycline in honey by high-performance liquid chromatography, Anal. Lett.,23(4), 607-616 (1990)13.Bonta V., Mrghita L.A., Dezmirean D., Moise A., Bobi O. and Maghear O., Optimization of HPLC method for quantifying tetracycline residue in honey, Bull. Uni. Agri. Sci. Veter. Med.,63/64, 186-190 (2007)14.Li J., Chen L., Wang X., Jin H., Ding L., Zhang K. and Zhang H., Determination of tetracyclines residues in honey by on-line solid-phase extraction high-performance liquid chromatography, Talanta,75(5), 1245-1252 (2008)15.Hakuta T., Shinzawa H. and Ozaki Y., Practical method for the detection of tetracyclines in honey by HPLC and derivative UV-Vis spectra, Anal. Sci., 25 (9), 1149-1153 (2009)16.Hai Y.Z., Dong H.C., Yi Z. and Hong T.H., Analysis of tetracyclines (TCs) residues in honey with HPLC – UV, Advan. Mat. Res.,159, 89-94 (2010)17.Anna G., Andrzej P., Andrzej B., Tomasz B. and Jan ., Oxytetracycline residues in honey analyzed by liquid chromatography with UV detection, J. Api. Sci.,57 (1), 25-31 (2013)18.Argauer R.J. and Moats W.A., Degradation of oxytetracycline in honey as measured by fluorescence and liquid chromatographic assays, Apidologie, 22(2), 109 – 115 (1991)19.Pena A., Pelantova N., Lino C.M., Silveira M.I.N. and Solich P., Validation of an analytical methodology for determination of oxytetracycline and tetracycline residues in honey by HPLC with fluorescence detection, J. Agri. Food Chem.,53(10), 3784-3788 (2005)20.Narin T. and Supaporn S., Determination of tetracycline antibiotic residues in honey samples collected from northern places of Thailand by HPLC, KMITL Sci. J.,8(2), 18-25 (2008) 21.Supaporn S. and Narin T., HPLC-Fluorescence detection method for quantitative determination of tetracycline antibiotic residues in honey, Naresuan Uni. J.,6(2), 147-155 (2009)22.Sun X., He X., Zhang Y. and Chen L., Determination of tetracyclines in food samples by molecularly imprinted monolithic column coupling with high performance liquid chromatography, Talanta,79 (3), 926-934 (2009)23.Ishii R., Horie M., Murayama M. and Maitani T., Analysis of tetracyclines in honey and royal jelly by LC/MS/MS, Shokuhin Eiseigaku Zasshi,47(6), 277-283 (2006)24.Carrasco P.A., Casado T.S., Segura C.A. and Fernández G.A., Reversed-phase high-performance liquid chromatography coupled to ultraviolet and electrospray time-of-flight mass spectrometry on-line detection for the separation of eight tetracyclines in honey samples, J. Chromatogr. A, 1195(1-2), 107-116 (2008)25.Jing T., Gao X.D., Wang P., Wang Y., Lin Y.F., Hu X.Z., Hao Q.L., Zhou Y.K. and Mei S.R., Determination of trace tetracycline antibiotics in foodstuffs by liquid chromatography-tandem mass spectrometry coupled with selective molecular-imprinted solid-phase extraction, Anal. Bioanal. Chem., 393(8), 2009-2018 (2009)26.Mei B., Rong L., Tingting C., Shouhui D., Hualin Z., Shuming Y. and Jing Q., Simultaneous determination of tetracycline antibiotics in beehives by liquid chromatography–triple quadrupole mass spectrometry, Advan. Appl. Sci. Res.,3(1), 462-468 (2012)27.Huang H.N., Chen T.B., Chen R.M. and Rao P.F., Detection of residual antibiotics in honey by capillary high performance liquid chromatography, Se Pu (Chinese Journal of Chromatography), 17(6), 588-589 (1999)28.Imdad U.M.Z., Khaliqur R., Arshad H. and Shafqatullah., Detection and quantification of antibiotics residues in honey samples by chromatographic techniques, Middle-East J. Sci. Res., 14(5), 683-687 (2013)29.Chen T.B., Deng W.H., Lu W.H., Chen R.M. and Rao P.F., Detection of residual antibiotics in honey with capillary electrophoresis, Se Pu (Chinese Journal of Chromatography), 19(1), 91-93 (2001)30.Wim R., Sigrid O., Hubert D.B. and Els D., Validation of the tetrasensor honey test kit for the screening of tetracyclines in honey, J. Agri. Food Chem.,55(21), 8359–8366 (2007)31.Casado T.S., Segura C.A., Busi S., Dinelli G. and Fernández G.A., Determination of tetracycline residues in honey by CZE with ultraviolet absorbance detection, Electrophoresis, 28(16), 2882-2887 (2007) Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 59-65, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 65 32.Wish T., Senee K., Richard L.D., Boonsom L., Sunantha W. and Saisunee L., Sequential injection spectrophotometric determination of tetracycline antibiotics in pharmaceutical preparations and their residues in honey and milk samples using yttrium (III) and cationic surfactant, Talanta, 84(5), 1401– 1409 (2011)33.Pagliuca G., Gazzotti T., Serra G. and Sabatini A.G., A scientific note on the determination of oxytetracycline residues in honey by high-performance liquid chromatography with UV detection, Apidologie, 3(6), 583 – 584 (2002)34.European Union-MRLs, Regulation (EC) No 396/2005, MRLs updated on 06/06/2013. A database of EU MRLs and pesticide authorization status (Dir 91/414/EEC), (2013)