Development and validation of determinative and confirmatory LC–MS/MS methodologies for total florfenicol and tulathromycin residues in bovine, equine and porcine kidney, liver and muscle tissues

•High matrix suppression is repeatable across tissue type and species.•Variance due to equipment repeatability contributes to overall method variance.•Deuterated analog versus non-analog internal standard correction can be comparable.

Separate methods for the quantitation and confirmation of regulatory relevant residue concentrations of total florfenicol and tulathromycin residues in multiple tissue matrices were developed and validated. Total florfenicol residues, determined and expressed as florfenicol amine (FFA) equivalents, were quantified and confirmed over a concentration range of 100–4000 ng/g, with an LOD of 33 ng/g, while total tulathromyicn residues, determined as CP-60,300 and expressed as tulathromycin equivalents, were quantified and confirmed over a concentration range of 500–10,000 ng/g, with an LOD of 300 ng/g. A 2 or 1 h acid digestion for the FFA and tulathromycin methods, respectively, followed by extraction, cleanup, and concentration using mixed-mode strong cation-exchange SPE cartridges was used. Quantitation and confirmation were accomplished using commercially available tri-deuterated FFA (FFA-D3) as internal standard and multiple reaction monitoring (MRM) of three transitions per target analyte. Mean recoveries and matrix effects were 60% and 25%; and 100% and 110%, respectively for the target analytes florfenicol amine and CP-60,300. Intra-lab method reproducibilities ranged from 7 to 11% RSD, which are within the AOAC recommended HORRATr guidelines for method reproducibilities estimated from single laboratory validation studies. Blind spikes showed that method bias was generally less than 15% for both methods within the calibration range. Both methods have been shown to meet requirements for use in national chemical residue monitoring programs.