Development of LC-MS/MS methodology for the detection/determination and confirmation of chloramphenicol, chloramphenicol 3-O-ß-d-glucuronide, florfenicol, florfenicol amine and thiamphenicol residues in bovine, equine and porcine liver.

A method for the detection and confirmation of organic solvent extractable residues of the neutral, acidic, and basic analytes of the amphenicol class veterinary drugs and selected metabolites was developed and validated. Using a modified QuEChERS extraction with SPE cleanup and LC-MS/MS analysis, limits of detection and confirmation for the different analytes in bovine, equine, and porcine liver ranged from 0.1ng/g for chloramphenicol to 1ng/g for florfenicol amine. Tissue homogenization with an ammonium formate/EDTA solution and subsequent analyte partitioning against 7:3 acetonitrile:isopropanol solution and mixed-mode strong-cation exchange solid-phase extraction cartridge cleanup allowed for the extraction of all compounds from tissues with mean recoveries ranging from 50% (chloramphenicol 3-O-ß-d-glucuronide) to 90% (thiamphenicol). Matrix effects ranged from greater than 85% suppression for florfenicol amine to 70% matrix enhancement for chloramphenicol 3-O-ß-d-glucuronide. Quantitation and confirmation were accomplished using commercially available penta-deuterated chloramphenicol as internal standard and multiple reaction monitoring (MRM) of two or three transitions per target analyte. Method accuracy was greater than 15% for all compounds except the glucuronide metabolite. Intra-lab method repeatability estimates ranged from 73% RSD for chloramphenicol 3-O-ß-d-glucuronide to 14% RSD for chloramphenicol. Only chloramphenicol 3-O-ß-d-glucuronide and florfenicol amine at the low end of their calibration ranges (0.25 and 1ng/g, respectively) did not meet AOAC recommended HorRatr guidelines for intra-lab repeatabilities. Preliminary tests show that the method's extraction protocol can be used to recover analytes of the ß-agonists, corticosteroids, fluoroquinolones, sulfonamides, and tetracycline drug classes from the same matrices. Requirements for use in national chemical monitoring programs as a detection/confirmatory (florfenicol amine and chloramphenicol 3-O-ß-d-glucuronide) and determinative/confirmatory (chloramphenicol, florfenicol, thiamphenicol) analytical methodology are met.

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.

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 100-4000ng/g, with an LOD of 33ng/g, while total tulathromyicn residues, determined as CP-60,300 and expressed as tulathromycin equivalents, were quantified and confirmed over a concentration range of 500-10,000ng/g, with an LOD of 300ng/g. A 2 or 1h 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.