LC/MS/MS Assay for the Simultaneous Quantitation of Ionophore Antibiotic Residues in Animal Tissues and Manure

Dayue Shang*, Nicole Gibbons, Monica Dyck, Jagroop Dahiya, Xiaoyan Jia
Health Canada, HPFB, Organic Residues Laboratory, Burnaby, B.C., Canada

Abstract

Introduction
The ionophores are a group of antibiotics widely used in livestock production to prevent coccidiosis (in chickens, turkeys, and cattle ) and for growth promotion (in cattle, and swine). Suitable trace analytical methods are needed to monitor the presence of ionophore antibiotics in animal tissue and food, resulting from improper use or from feed cross-contamination, and to study the rate of these antibiotics released into the environment in feedlot manure. There is currently no method capable of determining ionophore residues in these matrices. The aim of this study was to develop a rapid method which could be used to examine a large number of tissue samples from different animal species, as well as cattle manure.

Preliminary Data
Traditionally, for "dirty" matrices like cattle tissue and manure, multiple extraction, solid phase extraction (SPE) clean-up, and concentration are needed before ionophore residues can be analyzed by HPLC. The problems with this approach are that it is labour intensive and often results in poor reproducibility of the method. Taking advantage of the superior specificity of MRM mode in mass spectrometry, we developed a simple extraction process with no SPE clean-up, which significantly reduced the sample preparation time and yield acceptable recoveries. Matrix effects were dealt with by using internal standards, LC column focussing and matrix match calibration standards. Addition of EDTA in the mobile phase helped to improve method reproducibility, as all these compounds formed sodium adducts. Validation data are presented. Spike recovery was over 60% for all four ionophores (lasalocid, monensin salinomycin and narasin) with relative standard deviations of 4 to 15% for the compounds. The low quantitation limit for all the ionophores was 0.5 ppb (ng/g) and high limit of quantitation 100 ppb. The method sample turn out was 21 real world samples per day for one chemist. A small survey was conducted using this newly developed method.