Normalization strategies for metabonomic analysis of urine samples

Unlike plasma and most biological fluids which have solute concentrations that are tightly controlled, urine volume can vary widely based upon water consumption and other physiological factors. As a result, the concentrations of endogenous metabolites in urine vary widely and normalizing for these effects is necessary. Normalization approaches that utilized urine volume, osmolality, creatinine concentration, and components that are common to all samples (“total useful MS signal”) were compared in order to determine which strategies could be successfully used to differentiate between dose groups based upon the complete endogenous metabolite profile. Variability observed in LC/MS results obtained from targeted and non-targeted metabonomic analyses was highly dependent on the strategy used for normalization. We therefore recommend the use of two different normalization techniques in order to facilitate detection of statistically significant changes in the endogenous metabolite profile when working with urine samples.