title: Metabolomic analysis of urine samples by UHPLC-QTOF-MS: Impact of normalization strategies.
abstract: Among the various biological matrices used in metabolomics, urine is a biofluid of major interest because of its non-invasive collection and its availability in large quantities. However, significant sources of variability in urine metabolomics based on UHPLC-MS are related to the analytical drift and variation of the sample concentration, thus requiring normalization. A sequential normalization strategy was developed to remove these detrimental effects, including: (i) pre-acquisition sample normalization by individual dilution factors to narrow the concentration range and to standardize the analytical conditions, (ii) post-acquisition data normalization by quality control-based robust LOESS signal correction (QC-RLSC) to correct for potential analytical drift, and (iii) post-acquisition data normalization by MS total useful signal (MSTUS) or probabilistic quotient normalization (PQN) to prevent the impact of concentration variability. This generic strategy was performed with urine samples from healthy individuals and was further implemented in the context of a clinical study to detect alterations in urine metabolomic profiles due to kidney failure. In the case of kidney failure, the relation between creatinine/osmolality and the sample concentration is modified, and relying only on these measurements for normalization could be highly detrimental. The sequential normalization strategy was demonstrated to significantly improve patient stratification by decreasing the unwanted variability and thus enhancing data quality.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
authors.forename: Yoric
authors.initials: Y
authors.lastname: Gagnebin
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Tonoli
authors.affiliation: Department of Genetic and Laboratory Medicine, Geneva University Hospitals (HUG), Geneva, Switzerland.
authors.forename: Pierre
authors.initials: P
authors.lastname: Lescuyer
authors.affiliation: Service of Nephrology, Geneva University Hospitals (HUG), Geneva, Switzerland.
authors.forename: Belen
authors.initials: B
authors.lastname: Ponte
authors.affiliation: Service of Nephrology, Geneva University Hospitals (HUG), Geneva, Switzerland.
authors.forename: Sophie
authors.initials: S
authors.lastname: de Seigneux
authors.affiliation: Service of Nephrology, Geneva University Hospitals (HUG), Geneva, Switzerland.
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Martin
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
authors.forename: Julie
authors.initials: J
authors.lastname: Schappler
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland.
authors.forename: Julien
authors.initials: J
authors.lastname: Boccard
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland. Electronic address: serge.rudaz@unige.ch.
authors.forename: Serge
authors.initials: S
authors.lastname: Rudaz
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003404:Creatinine; D006801:Humans; D013058:Mass Spectrometry; D055432:Metabolomics; D009994:Osmolar Concentration; D051437:Renal Insufficiency / Q000175:diagnosis; D016482:Urinalysis / Q000379:methods*; D014556:Urine / Q000737:chemistry*
chemical_list: D003404:Creatinine
keywords: Kidney failure; LC-MS; Metabolomics; Normalization; Urine