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 blood 10 samples is not readily available in veterinary diagnostic laboratories despite being routine in 11 human clinical laboratories. A method utilizing HPLC-FLD instrumentation will be extrapolated 12 from an HPLC method validated in fish tissues and roe into an HPLC-FLD method for increased 13 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 lead 15 laboratory using prepared and verified homogeneous test materials including animal and pet 16 feed, animal tissues (liver, brain, and muscle), blood, and three species of fish roe. A Level 3 17 FDA validation will then be done by collaborators at the Cornell University Animal Health 18 Diagnostic Center and supply additional recommendations for optimization of the method. After 19 completion of the validation, the SOP for the Thiamine HPLC-FLD quantification method will be 20 made available to Vet-LIRN network laboratories and added to the literature in a peer-reviewed 21 journal. Oral and/or poster presentations at appropriate conferences will be made to increase 22 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/VIS 24 to increase accessibility in Vet-LIRN laboratories without HPLC-FLD testing capabilities in case 25 of an event requiring significant surge analysis capacity.