Rapid analysis of aminoglycoside antibiotics in bovine tissues using disposable pipette extraction and ultrahigh performance liquid chromatography–tandem mass spectrometry

Abstract

A high-throughput qualitative screening and identification method for 9 aminoglycosides of regulatory interest has been developed, validated, and implemented for bovine kidney, liver, and muscle tissues. The method involves extraction at previously validated conditions, cleanup using disposable pipette extraction, and analysis by a 3min ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method. The drug analytes include neomycin, streptomycin, dihydrosptreptomycin, and spectinomycin, which have residue tolerances in bovine in the US, and kanamicin, gentamicin, apramycin, amikacin, and hygromycin, which do not have US tolerances established in bovine tissues. Tobramycin was used as an internal standard. An additional drug, paromomycin also was validated in the method, but it was dropped during implementation due to conversion of neomycin into paromomycin. Proposed fragmentation patterns for the monitored ions of each analyte were elucidated with the aid of high resolution MS using a quadrupole-time-of-flight instrument. Recoveries from spiking experiments at regulatory levels of concern showed that all analytes averaged 70–120% recoveries in all tissues, except hygromycin averaged 61% recovery. Lowest calibrated levels were as low as 0.005μg/g in matrix extracts, which approximately corresponded to the limit of detection for screening purposes. Drug identifications at levels <0.05μg/g were made in spiked and/or real samples for all analytes and tissues tested. Analyses of 60 samples from 20 slaughtered cattle previously screened positive for aminoglycosides showed that this method worked well in practice. The UHPLC–MS/MS method has several advantages compared to the previous microbial inhibition screening assay, especially for distinguishing individual drugs from a mixture and improving identification of gentamicin in tissue samples.