Validation of quantative method for thiamine detection in animal feed tissue and plasma

1 Project Summary 2 The Thiamine HPLC-FLD quantification project is proposed by the WADDL-ASL laboratories to 3 fill the analytical gap in the Vet-LIRN network by developing a standardized HPLC-FLD 4 (fluorescence) method to quantify the concentration of thiamine thiamine 5 pyrophosphate(diphosphate) and thiamine monophosphate in animal feeds tissues and blood 6 that can be implemented by all in-network laboratories with HPLC-FLD capabilities. Thiamine is 7 an essential vitamin that must be supplied in the diet of many animal species and severe 8 disease can quickly develop in deficient diets. The current gold standard for diagnosis relies on 9 recognition of clinical signs and response to treatment as thiamine testing in tissue and blood10 samples is not readily available in veterinary diagnostic laboratories despite being routine in11 human clinical laboratories. A method utilizing HPLC-FLD instrumentation will be extrapolated12 from an HPLC method validated in fish tissues and roe into an HPLC-FLD method for increased13 sensitivity and the matrices expanded to include animal and pet feed and animal (non-fish)14 tissues (liver brain and muscle) and blood. A Level 2 FDA validation will be done in the lead15 laboratory using prepared and verified homogeneous test materials including animal and pet16 feed animal tissues (liver brain and muscle) blood and three species of fish roe. A Level 317 FDA validation will then be done by collaborators at the Cornell University Animal Health18 Diagnostic Center and supply additional recommendations for optimization of the method. After19 completion of the validation the SOP for the Thiamine HPLC-FLD quantification method will be20 made available to Vet-LIRN network laboratories and added to the literature in a peer-reviewed21 journal. Oral and/or poster presentations at appropriate conferences will be made to increase22 visibility and awareness of the new standardized SOP available for use and implementation.23 Additionally method development will be completed to expand the method to an HPLC-UV/VIS24 to increase accessibility in Vet-LIRN laboratories without HPLC-FLD testing capabilities in case25 of an event requiring significant surge analysis capacity.