Determination of Five Macrolide Antibiotic Residues in Raw Milk Using Liquid Chromatography−Electrospray Ionization Tandem Mass Spectrometry

A confirmatory method using liquid chromatography−electrospray ionization tandem mass spectrometry for determination of five macrolide antibiotics including spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin in raw milk is presented. Macrolides were extracted from raw milk by acetonitrile, and sample extracts were further cleaned up using solid-phase extraction cartridges. Data acquisition was achieved using multiple reaction monitoring, that is, two transitions, to provide a high degree of sensitivity and specificity. Matrix-matched standard calibration curves with the use of roxithromycin as an internal standard were utilized to achieve the best accuracy of the method. Both a conventional validation procedure and a designed experiment were applied to study the accuracy and precision of the method. The measurement uncertainty arising from accuracy and precision was estimated. The method accuracy, expressed as a percentage of overall recovery, was 100%, and its intermediate precision was <10%. LC-ESI/MS/MS method detection limits (S/N ≥ 3:1) of five macrolides were <0.3 μg/kg.

Keywords: Antibiotics; macrolides; raw milk; experimental design; measurement uncertainty; LC-ESI/MS/MS