title: Reproducibility of NMR analysis of urine samples: impact of sample preparation, storage conditions, and animal health status.
abstract: BACKGROUND
  Spectroscopic analysis of urine samples from laboratory animals can be used to predict the efficacy and side effects of drugs. This employs methods combining (1)H NMR spectroscopy with quantification of biomarkers or with multivariate data analysis. The most critical steps in data evaluation are analytical reproducibility of NMR data (collection, storage, and processing) and the health status of the animals, which may influence urine pH and osmolarity.
  
  
  METHODS
  We treated rats with a solvent, a diuretic, or a nephrotoxicant and collected urine samples. Samples were titrated to pH 3 to 9, or salt concentrations increased up to 20-fold. The effects of storage conditions and freeze-thaw cycles were monitored. Selected metabolites and multivariate data analysis were evaluated after (1)H NMR spectroscopy.
  
  
  RESULTS
  We showed that variation of pH from 3 to 9 and increases in osmolarity up to 6-fold had no effect on the quantification of the metabolites or on multivariate data analysis. Storage led to changes after 14 days at 4°C or after 12 months at -20°C, independent of sample composition. Multiple freeze-thaw cycles did not affect data analysis.
  
  
  CONCLUSIONS
  Reproducibility of NMR measurements is not dependent on sample composition under physiological or pathological conditions.
authors.affiliation: numares GROUP, Josef Engert Straße 9, 93053 Regensburg, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Schreier
authors.forename: Werner
authors.initials: W
authors.lastname: Kremer
authors.forename: Fritz
authors.initials: F
authors.lastname: Huber
authors.forename: Sindy
authors.initials: S
authors.lastname: Neumann
authors.forename: Philipp
authors.initials: P
authors.lastname: Pagel
authors.forename: Kai
authors.initials: K
authors.lastname: Lienemann
authors.forename: Sabine
authors.initials: S
authors.lastname: Pestel
mesh_terms: D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D015925:Cryopreservation; D005260:Female; D005615:Freezing; D005665:Furosemide / Q000494:pharmacology; D006304:Health Status; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D055442:Metabolome / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012965:Sodium Chloride / Q000494:pharmacology; D013048:Specimen Handling / Q000379:methods*; D014556:Urine / Q000737:chemistry*
chemical_list: D002070:Butadienes; D012965:Sodium Chloride; D005665:Furosemide; C001335:hexachlorobutadiene