A rapid and simple multi-residue methodology for quantification of anthelmintic macrocyclic lactones in cow, sheep and goat milk by HPLC: development, validation and application for the real samples

Abstract

Anthelmintic macrocyclic lactones (MLs) [ivermectin (IVM), abamectin (ABA), doramectin (DRM), eprinomectin (EPR) and moxidectin (MXD)] are used worldwide for the treatment of parasitic infections in large and small ruminants because of their low mammalian toxicity and higher efficacy against internal and external parasites at low dose levels. A rapid, simple and sensitive HPLC and a modified QuEChERS extraction method were developed and validated for the analysis of five anthelmintic MLs residues in cow, sheep and goat milk. The analytes were extracted from milk samples by acetonitrile deproteinization, followed by the addition of sodium sulphate and sodium chloride to generate a phase partition. HPLC separation was conducted at 50⁰C using a C18 analytical column (Zorbax, Eclipse Plus, 250 mm × 4,6 mm, 5µ). The mobile phase consisted of a mixture of acetonitrile: methanol: water (58:38:4, v/v/v), delivered by an isocratic program at the flow rate of 1.3 ml/min. The elution of five analytes was completed within 11 min. The fluorescence detector (FLD) was set at an excitation wavelength of 375 nm and an emission wavelength of 470 nm. Recoveries ranged between 78.44% and 92.66%. The quantification limits (LOQ) of anthelmintic MLs in the milk samples from 3 different ruminant species ranged between 2.01 and 3.10 ng/g. The developed method was validated in accordance with the criteria of Commission Decision 2002/657/EC. Linearity, decision limit, detection and quantification limits, recovery, accuracy, precision, sensitivity and selectivity were determined, and satisfactory results were obtained. This developed and validated method is rapid, simple, sensitive and environmentally friendly, and can be used for multiple residue analyses of anthelmintic MLs in cow, sheep and goat milk for human consumption. The quantification limits for all drugs were below the maximum residue limits (MRLs), making the method appropriate for routine testing. Finally, the application of this method was assessed using real samples of cow, sheep and goat milk. The presence of MLs was confirmed in 8 out of 70 tested milk samples, which shows the method can be successfully used to determine the residue of anthelmintic MLs in three types of milk.