Ion spray liquid chromatography/tandem mass spectrometry for the confirmation of aminoglycoside antibiotic residues in bovine kidney

In order to regulate aminoglycoside antibiotic residues in edible tissues of livestock, the FDA requires confirmatory methods based on mass spectrometry (MS). In this work a method is presented for the multi-residue confirmation of the following 6 priority aminoglycosides in bovine kidney: spectinomycin, hygromycin B, streptomycin, dihydrostreptomycin, gentamicin C complex, and neomycin B. FDA levels of concern in bovine kidney range from 0.02 ppm for hygromycin B to 0.5 ppm for streptomycin and dihydrostreptomycin. Because of the thermal lability and/or nonvolatility of these compounds ion spray HPLC/MS/MS was investigated.; Ion spray mass spectra of all compounds exhibited little fragmentation and base peaks were doubly-protonated doubly-charged molecules except for spectinomycin, which was singly-charged. HPLC/MS conditions were optimized with respect to ion-pairing agent, its concentration, and type of organic modifier. A 17-min gradient separation was developed with mobile phases consisting of aqueous 20 mM pentafluoropropionic acid and acetonitrile using a 2-mm i.d. YMCbasic column at a flow of 0.2 mL/min. With several modifications, an existing matrix solid-phase dispersion (MSPD) method was employed for tissue extraction. Protonated molecules served as precursor ions for collision-induced dissociation (CID) and 3 product ions were selected for selected reaction monitoring (SRM) where possible.; Validation of the confirmatory method was conducted through SRM HPLC/MS/MS analysis of negative control, fortified and incurred bovine kidney samples on two different days. All of the samples could be confirmed with ion ratios within 15% of the daily mean of fortified standards and 90% of the samples had ion ratios within 10%. Absolute retention time data of samples were all within 1% of a consecutively injected fortified standard except for dihydrostreptomycin, which displayed 3% variability. Use of retention time relative to the internal standard tobramycin provides for even better agreement in all cases except dihydrostreptomycin. All compounds except spectinomycin could be detected (while monitoring 3 ions by SRM) in bovine kidney tissue at or below the level of concern. MSPD recoveries were acceptable with the exception of the 27% value observed for spectinomycin.