A high-throughput UHPLC method for the analysis of 5-nitroimidazole residues in milk based on salting-out assisted liquid-liquid extraction

A novel salting-out assisted liquid-liquid extraction (SALLE) method has been developed for the extraction of eight 5-NDZ antibiotics from milk samples prior to their analysis by UHPLC. An exhaustive study of the parameters involved in the SALLE procedure has been carried out, optimizing the extraction solvent volume, the amount of the salt agent, and the centrifugation and vortex timing by an experimental design. After sample treatment, the obtained extract was reconstituted in the mobile phase (6:94 (v/v) acetonitrile/water containing 0.1% (v/v) of formic acid) and analyzed by the proposed UHPLC-UV method. Separation was accomplished in a C18 Zorbax Eclipse Plus (50 mm × 2.1 mm, 1.8 μm) column at 45 °C within 8 min. Gradient mode was considered using a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.45 mL/min. Analytical signals were monitored at 320 nm. Matrix-matched calibration curves showed satisfactory linearity (R2 ≥ 0.996). LODs, ranging from 2 to 4 μg/L, were achieved and precision studies showed RSDs lower than 12.8% in terms of peak height. Additionally, recoveries higher than 62.8% were obtained for all studied compounds in milk samples.