A multi-laboratory validation of the extraction of fat-soluble vitamins from tissue and feed and their analysis by LC-MS/MS

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and may utilize outdated equipment.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.

Grant Number: 1U18FD008026-01
NIH Institute/Center: FDA
Principal Investigator: John Buchweitz