Methodological validations of metabonomic analysis are far from being perfect.This research tends to validate the possibility of Quadrupole linear ion trap (QTRAP) developing into a promising tool in metabonomic studies from the perspective of methodological considerations.As an instance, a metabolomic profiling study on hyperlipidemia samples was conducted to certificate the conceptions.The metabonomic analysis was based on a HPLC-QTRAP method.Positive and negative ESI scanning modes were tentatively merged to shorten analysis time for an individual sample.Internal standards were applied in intra-day and inter-day analytical performance to verify the possibility of reducing variance.HPLC-QTRAP based methods for the analysis of human urine and plasma for metabonomic studies were built.The analytical protocol was applied in a hyperlipidemia metabonomic study.Multivariate statistical analysis methods were used to identify the metabolite profiles of hyperlipidemia and found some potential biomarkers.The results showed that intra-day relative stand deviations (RSDs) of 80% of reference standards which calculated by internal and external methods were less than 20%.However, most of the inter-day RSDs calculated with the same methods were comparably more than 20%.The metabolite profiles of hyperlipidemia was analyzed and some potential biomarkers, including citric acid, adenosine 2', 3'-cyclic phosphate, proline, 5-methyclcytosine, Lyso-PC (16∶0) and 3-methyluridine were identified.HPLC-QTRAP based metabonomics is a promising tool to find and identify potential biomarkers for metabonomic studies.