PAR-18-604 Vet-LIRN Network Capacity-Building Project A multi-laboratory validation of the extraction of fat-soluble vitamins from tissue and feed and their analysis by LC-MS/MS Abstract The fat-soluble vitamins A, E, and D are essential dietary components for all animal species. Although required in small quantities, these compounds exert a multitude of metabolic and physiologic effects through all stages of an animal’s life. Because these vitamins or their precursors are naturally occurring, most free ranging animals can maintain adequate levels through the browse or forage they consume. However, processed grains and other ingredients used in the generation of commercial feeds for companion, production, and specialty species are challenged with the propensity of vitamins toward degradation. Accordingly, the commercial feed industry supplements diets with synthetic forms of these vitamins to provide adequate nutrient value. However, storage conditions and mixing errors can adversely impact the final concentration that is consumed. As a result, nutrient vitamin concentrations may be either inadequate and lead to clinical signs of deficiency or excessive and exhibit manifestations of toxicosis. Clinically, both deficiencies and toxicoses can be established ante-mortem through the measurement of serum vitamin A, E, or the vitamin D metabolite, 25-hydroxy vitamin D (25-OH D). For the post-mortem animal, tissue vitamin analysis is generally performed with liver being the sample of choice for vitamins A and E, and kidney being the best sample for 25-OH D. A clinical diagnosis that is suggestive of vitamin deficiency or excess generally (but not always) implicates a potential problem with feed. Current methods to address fat soluble vitamin content in feed are somewhat onerous, time-consuming, and may utilize outdated equipment. We propose a method of extraction and analysis for both tissues and feed that is simple, accurate, and reproducible across laboratories within the CVM Vet-LIRN community, Michigan State University Veterinary Diagnostic Laboratory (MSU VDL), Kansas State University Veterinary Diagnostic Laboratory (KSU VDL), Tennessee Department of Agriculture Kord Animal Health Diagnostic Laboratory (TN Ag Lab), Texas A&M University Veterinary Medical Diagnostic Laboratory (TVMDL), University of California Davis California Animal Health and Food Safety (CAHFS) lab, and University of Guelph Animal Health Laboratory (Guelph AHL) equipped with liquid chromatography tandem mass spectrometry.

PAR-18-604 Vet-LIRN Network Capacity-Building Project A multi-laboratory validation of the extraction of fat-soluble vitamins from tissue and feed and their analysis by LC-MS/MS Project Narrative Although commercial feeds are supplemented with vitamins in known amounts that are guaranteed by the manufacturer, the net loss of vitamins during the manufacturing, distribution and storing processes may render the feed deficient for the animal’s overall needs (Riaz et al., 2009), and conversely, over-supplementation, especially with vitamin D or its 25-hydroxy metabolite, may render a feed as adulterated and potentially toxic. With either deficiency or excess, in the absence of routine monitoring of feed, animal serum or tissue serve as an initial diagnostic within veterinary diagnostic laboratories with the potential to trigger subsequent investigations into diet. Herein, we propose a two-phased approach to a multi-laboratory validation of a single analytical method for the extraction and quantitation of fat-soluble vitamins in both tissue and feed by LC- MS/MS.