Validation of HPLC method for determination of tetracycline residues in chicken meat and liver

High-performance liquid chromatographic method with diode-array detection (HPLC–DAD) was optimised and validated for determination of tetracyclines (TCs) residue in chicken meat and liver through evaluating each step of various methods. The principle steps involved ultrasonic-assisted extraction of TCs from chicken samples by 2 ml of 20% trichloroacetic acid and citrate buffer (pH 4) which gave a clearer supernatant and high recovery, followed by centrifugation and purification on SPE (Strata C18-E cartridge) using 10 ml of 0.01 M methanolic oxalic acid for TCs elution. Separation was on reversed-phase column (Nuclosil 100 C18, 25 cm × 4.6 mm ID, 5 μ) by multisteps gradient elution which provided a better chromatographic peak resolution and the late eluting peaks were as sharp as those eluting earlier. Monitoring was at 351 nm which gave a higher detector response factor. Validity study of the method revealed that all obtained calibration curves showed good linearity (r2 > 0.999) over the range of 50–5000 ng. Sensitivity was found to be 1.44, 1.90, 0.95 and 1.23 ng for OTC, TC, CTC and DC, respectively. Accuracy was in the range of 71.88–92.44.3% and 68.88–84.84% for meat and liver, respectively. Precision was lower than 10% in all cases indicating that the method can be used as a validated method. Limit of detection (LOD) was found to be 4.4, 5, 13 and 10 ng for OTC, TC, CTC and DC, respectively. The corresponding values of limit of quantitation (LOQ) were 10, 13, 27 and 22 ng.