Mixed micelle-cloud point extraction for the analysis of penicillin residues in bovine milk by high performance liquid chromatography

A mixed micelle-cloud point extraction (MM-CPE) has been developed for the analysis of penicillin antibiotics (ampicillin, penicillin G, oxacillin, and cloxacillin) in milk samples using Triton X-114 (TX-114) and cethyl trimethylammonium bromide (CTAB) as the mixed micellar extractant. The parameters affecting the MM-CPE that were investigated including solution pH, CTAB concentration, TX-114 concentration, electrolyte salt, equilibration temperature and incubation time. The optimum MM-CPE conditions were: 10 mmol L−1 phosphate buffer pH 8, 0.06% (w/v) CTAB, 1.5% (w/v) TX-114, and 7% (w/v) Na2SO4, and 5 min equilibration at 40 °C. The separation of penicillins was achieved within 8 min under the HPLC conditions: a Vydac C18 column, isocratic elution of 5 mmol L−1 phosphate buffer (pH 6.6) and methanol (55:45, v/v), and a flow rate of 1 mL min−1, with photodiode array detection at 215 and 244 nm. Under the selected condition, the proposed method gave linear calibrations in the range 0.002–10 μg mL−1 with correlation coefficients greater than 0.999. Limits of detection (LOD) were 2–3 ng mL−1, and 15–40-fold enhancement compared to that without preconcentration. Good reproducibility was achieved with relative standard deviation <5% for peak area and <3% for retention time. High accuracy, with recoveries higher than 80%, was obtained. The proposed mixed micelle-CPE-HPLC method has shown to be of high potential for the analysis of penicillin residues in milk with LOD comparable to the established maximum residue limits (4–30 ng mL−1).