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Two methods (LC-MS/MS and HPLC) for determination of ractopamine in animal tissues, and one method (LC-MS/MS) for monensin/narasin in animal tissues were adopted by an AOAC expert review panel (ERP), chaired by Leendert van Ginkel, RIKILT, as AOAC First Action Official MethodsSM, based on validation data generated by Elanco in the past 2 years. The methods had been previously recommended by ERPs for further evaluation and validation in 2009, from dozens of promising and potential candidate methods. The ERP, thoroughly vetted by the Official Methods Board, and with much of the same membership as in the original 2009 ERPs, evaluated methods for completeness of validation and likeliness of meeting fitness-for-purpose statements (appropriateness of intended use) in determining ractopamine and monensin/narasin in animal tissues. Priority compounds had previously been identified by a stakeholder panel in 2008. The method adopted by the ERP for determination and confirmation of parent and total ractopamine by liquid chromatography with tandem mass spectroscopy (LC-MS/MS) was validated in a single laboratory (SLV) for bovine, swine, and turkey tissues. The method uses methanol extraction of the tissues, followed by enzymatic hydrolysis if analyzing for total (parent plus conjugate) ractopamine. A mixed-mode cation exchange resin is used to clean up the initial extract before LC-MS/MS. Matrix-matched standards and a ractopamine-d6 internal standard are used for quantification of parent and total ractopamine in unknown samples. The HPLC method adopted for ractopamine was evaluated in an SLV study. The SLV study was carried out by Covance Laboratories (Greenfield, Indiana, USA) and included evaluation of linearity of standards, selectivity, recovery, bias, repeatability precision, and estimates of LOD and LOQ. Matrix studies were carried out with fortified and incurred tissues. In addition to method validation parameters, the stability of ractopamine in tissues, extracts, and standard solutions was examined. In addition, the ERP adopted an LC-MS/MS method for monensin/narasin. The validation was carried out by Charles River Laboratories (Edinburgh, UK) and included the determination of linearity of the standard curve, selectivity or the absence of interference from other compounds, repeatability precision in fortified and incurred tissues, recovery and bias in fortified tissues, LOD, LOQ, robustness of the method, and stability of the analyte in tissues, extracts, and matrix-matched standards. Fortified matrixes were prepared at approximately ½ MRL/tolerance, at the MRL/tolerance, and at 2x MRL/tolerance. In some cases, multiple MRL/tolerance values were accommodated by adding additional fortification levels. Incurred tissues were provided to the validating laboratory by Elanco Animal Health. Acceptance criteria for ractopamine, monensin, and narasin were based on those developed by the AOAC Stakeholder Panel for Veterinary Drug Residues. Overall, the goal is to update and modernize methodology that is fit-for-purpose, globally accepted, and meets regulatory testing requirements. The new ERP-approved methods have been codified (2011.22 Determination of Ractopamine in Swine, Bovine, and Turkey Tissues by HPLC with Fluorescence Detection; 2011.23 Determination and Confirmation of Parent and Total Ractopamine in Bovine, Swine, and Turkey Tissues by Liquid Chromatography with Tandem Mass Spectroscopy; 2011.24 Determination and Confirmation of Narasin and Monensin in Bovine, Swine, and Chicken Tissues by Liquid Chromatography with Tandem Mass Spectroscopy) and will be drafted into AOAC style for publication in the Journal of AOAC INTERNATIONAL and Official Methods of AnalysisSM. Full coverage is scheduled for the November/December 2011 issue of Inside Laboratory Management.
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