Figure 8: Effects of multiple freeze-thaw cycles. Glucose, citrate, lactate, creatinine, PAG, and urea were measured in urine samples from natrosol-, furosemide-, or HCBD-treated rats by 1H NMR quantification. Samples were stored at −20°C before and in-between the measurements ( per dot, urine samples pooled treatment-wise from two rats). They were thawed by a standardized procedure shortly before the measurements; the procedure was repeated for 5 freeze-thaw cycles. (a) Single metabolite quantification results are given as relative concentrations normalized to the first measurement. Values below LLOQ (i.e., glucose and lactate concentrations in the samples from natrosol- and furosemide-treated rats) were excluded from evaluation. The grey range (15%) and dotted horizontal lines (20%) show the range of variation stated as acceptable by the “Guidance for Industry” [12]. (b) Prediction of nephrotoxicity was performed using a metabonomics approach (ensemble classification system). For each sample a classifier value is given, labelling the given compound as “(non-)toxic”. The horizontal line at 0.5 is the limit for the prediction of nephrotoxicity, the dashed lines at 0.35 and 0.65 represent an “intermediate” range, related to the statistical significance of the prediction.