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Updated: Jun 25, 2025
Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS
Published on: May 20, 2013
Optimization and validation of metabolomics methods for feline urine and serum towards application in veterinary
Ellen Vanden Broecke1, Laurens Van Mulders1, Ellen De Paepe2
1Ghent University, Faculty of Veterinary Medicine, Department of Translational Physiology, Infectiology and Public Health, Laboratory of Integrative Metabolomics (LIMET), Salisburylaan 133, B-9820, Merelbeke, Belgium; Ghent University, Faculty of Veterinary Medicine, Department of Small Animals, Salisburylaan 133, B-9820, Merelbeke, Belgium.
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 (R2 ≥ 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 (R2(Y) > 0.95, Q2(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.
