Preanalytical standardization of amino acid and acylcarnitine metabolite profiling in human blood using tandem mass spectrometry

Quantitative metabolite profiling in biological samples has the potential to reflect physiological status and to identify disease associated disturbances in metabolic networks. However, this approach is hampered by a wide range of preanalytical variables. Hence, the aim of our study was to develop a standardized preanalytical protocol for metabolite profiling of amino acids and acylcarnitines in human blood. Amino acids and acylcarnitines were simultaneous analyzed after butylation of 3 mu L dried blood or 10 mu L whole blood, serum and anticoagulated plasma using electrospray tandem-mass spectrometry. The influence of exogenous and endogenous preanalytical variables was investigated in healthy volunteers. Different sampling materials and anticoagulants for blood taking were investigated. Concentrations of long-chain acylcarnitines were 5-fold higher in EDTA-whole blood or dried whole blood compared to serum and anticoagulated plasma. Significant differences in amino acid concentrations were found for capillary versus venous blood taking. Fasting for 8 h before specimen collection minimized the nutritional influence. Physical activity significantly alters amino acid and short chain acylcarnitine concentrations. As a result of our preanalytical investigation we developed a pre-treatment protocol based on EDTA whole blood dried on filter paper to reduce the preanalytical variability and facilitate reproducible quantitative metabolite profiling in clinical trials.