Macrocyclic lactone antibiotics such as ivermectin (IVM) belonging to the avermectin group, and moxidectin (MOX), a milbemycin, are commonly used to treat endo- and ectoparasite infestations in farm animals. Although they are relatively safe to use in mammals, reports of toxicity do exist. Therefore, the European Union has placed maximum residue limits (MRL) permissible in tissues destined for human consumption. There is also EU legislation in place to monitor animal feeds and limit food chain contamination.
Currently, food safety testing for lactone residues involves HPLC with UV detection. It is easy and rapid but is not sensitive enough for measuring low-level concentrations.
Galarini et al.1 report validation of a new, more sensitive method for quantifying lactone residues in tissues, milk and animal foodstuffs. Although methods that use sensitive liquid chromatography (LC) coupled with tandem mass spectrometric detection exist, the authors chose to validate a simpler method that uses easily accessible high performance liquid chromatography (HPLC) coupled with fluorescence detection.
The researchers set out to develop a multi-analyte protocol for simultaneously measuring different species of lactones in muscle, milk and feed-stuff samples using EU Commission Decision 2002/657/EC and ISO/IEC 17025 (2005) as guides. They used Spectrasystem (Thermo Scientific) chromatographic instrumentation for assay development. This comprises a P4000 quaternary pump, an AS3000 autosampler and a FL3000 fluorescence detector.
Galarini et al. used the following animal tissue samples in their testing matrices: liver (cow, chicken, sheep); muscle (cow, chicken and pig); and milk (cow, sheep). Animal feed samples for cow, pig and fish were also analyzed. The researchers added milbemycin A4 as the internal standard in each sample, and prepared dilutions from commercial grade stocks of abamectin (ABA), doramectin (DOR), emamectin (EMA), eprinomectin (EPR), IVM and MOX for assay calibration. They also used these stock dilutions to spike the test matrices, adding all six analytes to each sample.
After homogenization and spiking with test analytes and the internal standard, the researchers extracted lactones from the tissue/milk samples using acetonitrile and acetone for the feeds. They then derivatized the extracted lactones prior to HPLC analysis according to a method described by Danaher et al.2 Alongside each HPLC run, standard dilutions of the test analytes were injected at five different concentration points for calibration. Each test sample took 14 minutes to run. Fluorescence was detected at 364 nm excitation and 470 nm emission wavelengths.
Initial validation results showed good linearity between concentrations of 10 to 400 ngml-1 and also for analyte calibration curves (R2>0.998, deviations <10%). Comparison of assay results between laboratories was also consistent. Inter-laboratory testing in accordance with the Food Analysis Performance Scheme returned Z scores <1.5 at all times.
The researchers also found that by replacing acetic anhydride with trifluoroacetic anhydride in the derivation step, they could reduce preparation time since subsequent clean up prior to fluorescence detection was not needed.
The researchers calculated recovery of analytes spiked into each sample matrix (ranged 73% to 110%) and repeatability of the assay (CV ranged 1.7% to 20%). By investigating alternative extraction steps, they were able to improve assay precision for specific lactones. For example, EMA measurement was more accurate when the researchers used an alkaline solvent during extraction.
The EU directives controlling lactone contamination levels specify different MRLs for each analyte. Food safety testing must take this into consideration in addition to the differing MRLs in each animal species. In order to encompass this broad range of MRLs in their assay validation, Galarini et al. used a range of concentrations chosen to reflect the directives to spike each food matrix sample. In this way they demonstrated that the working range of the assay, 3.2 – 320 μg kg−1 was suitable for testing lactone MRLs in tissues and feeds according to EU food safety testing legislation.
The authors consider that their assay is a simple, robust and accurate method for checking for veterinary lactone MRLs in animal tissues and milk for human consumption, and in animal feeds. It is sensitive enough to comply with relevant food safety testing legislation within Italy and the EU.
1. Galarini, R. et al (2013) “Determination of macrocyclic lactones in food and feed“, Food Additives & Contaminants: Part A, Vol. 30, No. 6, (pp.1068–1079), http://dx.doi.org/10.1080/19440049.2013.781275
2. Danaher, M. et al. (2001) “Development and optimisation of an improved derivatization procedure for the determination of avermectins and milbemycins in bovine liver“, Analyst 126 (pp.576–580)
Further Reading – Spectrasystem Instrumentation
- AS300 autosampler product manual
- FL3000 Fluorescence Detector product manual
- P4000 Quaternary Pump product manual