A confirmatory and quantitative method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed for simultaneous determination of 13 aminoglycoside antibiotics in various samples. The aminoglycoside analytes were released and extracted from different matrices with 5% trichloroacetic acid. The influence of pH values on the solid-phase exaction (SPE) procedure has been studied. Due to different pK(a) values of the compounds, seven aminoglycosides (AGs) were quantitatively retained on Oasis HLB cartridges at pH<1 and then six aminoglycosides were retained at pH 8.5. Thus, the combination of two HLB SPE cartridges with different pH values was involved to simultaneously purify 13 aminoglycosides. The proposed two SPE steps produced high recovery yields for every aminoglycoside in five different matrices. The LC-MS/MS method was validated according to the European Union Commission directive 2002/657/EC. Good performance characteristics were obtained for recovery, precision, calibration curve, stability, specificity, decision limits (CCalpha) and detection capabilities (CCbeta) in different matrices. The optimized procedure has been successfully applied to real samples in our laboratories (n> or =200) for 1 year. It demonstrated that the new method was robust and useful for identification and quantification of 13 aminoglycosides residues in foods of animal origin.