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Optimal Eluting Condition for Analysis of Tetracycline and Its Derivatives in Shrimp using High Performance Liquid Chromatography Napawan Tumsangthong, Varipat Areekul and Tatiya Siharai A high- performance liquid chromatography with photodiode-ar ay detector (HPLC-DAD) was optimized for analysis of oxytetracycline, tetracycline and chlortetracycline in shrimp. The performance criteria was considered retention time (RT), resolution (Rs) and capacity factor (k') comparing between isocratic elution, modified isocratic elution and gradient elution. The RT, Rs and k were statistical y dif erent among elution conditions (p≤0.05). The result obtained from fortified sample indicated that the best condition for analysis was using the gradient elution which the separation time was accomplished at 9.2 min and no overlapping peak was observed. The recovery mean (%) was at the international acceptable level. Key Words: Tetracycline, HPLC, shrimp, optimal condition E-mail address: [email protected] (N. Tumsangthong) Faculty of Agro-Industry, King Mongkut s Institute of Technology Ladkrabang, Ladkrabang, Bangkok 10520 (Maximum Residue Limits, MRLs) F ก F ก Quality Control Division Department of Fisheries, 2006) (oxalic acid, AR grade, Fisher Scientific), (chlortetracycline hydrochloride CTC) 2. ก ZORBAX Eclipse XDB-C8, 250x 4.6 mm, 5 µm (Agilent) ก ก ก กก (resolution, Rs) กก F 2 F (capacity factor, k') กก F 2 (Synder and Kirkland, 1979) ก ก ก กก (resolution, Rs) F ก 1.04 Figure 1 Chromatogram of 0.6 mg/Kg standard mixture of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) (0.2 mg/kg of each standard) ; Isocratic elution condition (a), modified isocratic elution condition (b) and gradient elution condition (c) Table 1 Retention time, capacity factor and resolution of tetracycline and its derivatives a, b, c Mean with dif erent let ers within a row are statistical y significant dif erence (α =0.05) Figure 2 Chromatogram of sample blank (left) and fortified sample (right) with 0.6 mg/Kg standard mixture of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) (0.2 mg/kg of each standard) using dif erent elution; isocratic elution condition (a,b), modified isocratic elution (c,d) and gradient elution condition(e,f) Table 2 Recovery mean (%) of oxytetracycline (OTC) tetracycline (TC) and chlortetracycline (CTC) in fortified shrimp using gradient elution condition RSD: Relative Standard Deviation n = number of replicates (10) . 2534. Principles and techniques of instrumental analysis. F F F Cinquina, A.L., F. Longo, G. Anastasi, L. Giannet i and R. Cozzani. 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, 227-233. Codex Alimentarius Commission. 2002. Codex commit ee on residues of veterinary drugs in Fish Inspection and Quality Control Division Department of Fisheries, Thailand : April 2006; Revision I I. Chemical Reference Criteria for Fishery Product. Snyder, L.R. and J.J. Kirkland. 1979. Introduction to modern liquid chromatography. 2nd Ed. New

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