Optimization and validation of metabolomics methods for feline urine and serum towards application in veterinary medicine.

Background: Metabolomics is an emerging and powerful technology that offers a comprehensive view of an organism's physiological status. Although widely applied in human medicine, it is only recently making its introduction in veterinary medicine. As a result, validated metabolomics protocols in feline medicine are lacking at the moment. Since biological interpretation of metabolomics data can be misled by the extraction method used, species and matrix-specific optimized and validated metabolomic protocols are sorely needed.

Results: Systematic optimization was performed using fractional factorial experiments for both serum (n = 57) and urine (n = 24), evaluating dilution for both matrices, and aliquot and solvent volume, protein precipitation time and temperature for serum. For the targeted (n = 76) and untargeted (n = 1949) validation of serum respectively, excellent instrumental, intra-assay and inter-day precision were observed (CV ≤ 15% or 30%, respectively). Linearity deemed sufficient both targeted and untargeted (R ≥ 0.99 or 0.90, respectively). An appropriate targeted recovery between 70 and 130% was achieved. For the targeted (n = 69) and untargeted (n = 2348) validation of the urinary protocol, excellent instrumental and intra-assay precision were obtained (CV ≤ 15% or 30%, respectively). Subsequently, the discriminative ability of our metabolomics methods was confirmed for feline chronic kidney disease (CKD) by univariate statistics (n = 41 significant metabolites for serum, and n = 55 for urine, p-value<0.05) and validated OPLS-DA models (R(Y) > 0.95, Q(Y) > 0.65, p-value<0.001 for both matrices).

Significance: This study is the first to present an optimized and validated wholistic metabolomics methods for feline serum and urine using ultra-high performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry. This robust methodology opens avenues for biomarker panel selection and a deeper understanding of feline CKD pathophysiology and other feline applications.