The author introduces an organic solvent-free, small-scale, time shortening, economical procedure for quantitation of oxytetracycline (OTC) and its 4-epimer, 4-epi-oxytetracycline (4eOTC), in cow’s milk. Sample preparation was performed by homogenization using a handheld ultrasonic-homogenizer with water followed by Mono Tip® C18 pipette tip contains silica monolith bonded with octadecyl group with water eluent. The HPLC separation was achieved a C4 column with an isocratic aqueous mobile phase and a photodiode array detector (PDAD). The total time required for the analysis of one sample was less than 6 min. The method validation data were well within the international analytical method acceptance criteria. The limits of quantitation for OTC and 4eOTC were 0.035 and 0.048 μg/mL, respectively. The present technique may be proposed as an international standardized analytical method for routine residue monitoring for OTC and 4eOTC in milk.
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