Biological variability dominates and influences analytical variance in HPLC-ECD studies of the human plasma metabolome

BACKGROUND: Biomarker-based assessments of biological samples are widespread in clinical, pre-clinical, and epidemiological investigations. We previously developed serum metabolomic profiles assessed by HPLC-separations coupled with coulometric array detection that can accurately identify ad libitum fed and caloric-restricted rats. These profiles are being adapted for human epidemiology studies, given the importance of energy balance in human disease. METHODS: Human plasma samples were biochemically analyzed using HPLC separations coupled with coulometric electrode array detection. RESULTS: We identified these markers/metabolites in human plasma, and then used them to determine which human samples represent blinded duplicates with 100% accuracy (N = 30 of 30). At least 47 of 61 metabolites tested were sufficiently stable for use even after 48 hours of exposure to shipping conditions. Stability of some metabolites differed between individuals (N = 10 at 0, 24, and 48 hours), suggesting the influence of some biological factors on parameters normally considered as analytical. CONCLUSION: Overall analytical precision (mean median CV, ~9%) and total between-person variation (median CV, ~50-70%) appear well suited to enable use of metabolomics markers in human clinical trials and epidemiological studies, including studies of the effect of caloric intake and balance on long-term cancer risk.