Development and validation of reverse - phase HPLC method for detection of tetracycline residue in fresh milk samples

Abstract

Dairy products are highly required by humans for growth, development and repairs as they are good sources of essential nutrients. Indiscriminate administration of veterinary drugs for prophylactic and therapeutic purposes could lead to the presence of their residues in dairy products such as milk. Reversed-phase high performance liquid chromatographic method with UV detection was developed and validated for detection of tetracycline in milk from five different samples. Mobile phase consisting of a mixture of deionized water, acetonitrile, monobasic potassium and acetic acid was delivered at a flow rate of 1.0 mL/min. The column used was Agilent ZORBAX ODS 4.6 X 150 mm 5μm.. The eluent was monitored by UV detection at 220 nm. Analysis was performed at 35oC and the total run time was 20 minutes. Analytical parameters such as linearity, accuracy, precision and limit of detection were determined by validation procedure and found to be satisfactory. Calibration curve was linear over the concentration range of 0.01- 0.5 mg/mL with an R² of 0.9978. The limits of detection and limit of quantification were found to be 0.00023 mg/mL and 0.00110 mg/mL, respectively. The % RSD is found to be 3.62, 0.23 and 0.44 for 0.01, 0.1 and 0.5 mg/mL concentration with percentage recovery of 78.7 – 98.6 %. Generally, the developed method was found to be simple, rapid, precise and accurate for quality control of tetracycline in fresh milk samples.