The biochemical metabolite screen in the Munich ENU Mouse Mutagenesis Project: determination of amino acids and acylcarnitines by tandem mass spectrometry

Background: Gene mutations often result in altered protein expression and, In turn, lead to changes in metabolite levels in one or more distinct bioch emical pathways. Traditional analytical methods for metabolite determinatio n are usually time consuming, expensive, and, thus, not suitable for high t hroughput analysis. However, recent developments in electrospray-tandem-mas s-spectrometry allow comprehensive metabolite scanning from very small amou nts of blood with high speed, cost effectiveness, and accuracy. Methods: A blood spot from a filter paper equivalent to 3 mu l of blood was punched ou t and transferred to a 96-well microtiter plate. After addition of a set of 14 stable isotope-labeled internal standards, amino acids and acylcarnitin es were extracted with methanol. The dried residue was derivatized with but anolic hydrochloric acid and subjected to MSMS analysis. Results: Acylcarni tines were all determined by a precursor ion scan of 85 Da. Neutral loss sc anning of 102 Da was suitable for the quantitation of threonine, serine, pr oline, histidine, alanine, aspartic acid, glutamic acid, methionine, tyrosi ne, phenylalanine, isoleucine/leucine and valine. Glycine was detected by a loss of a 56-Da fragment, whereas a 119-Da loss was suitable for the measu rement of citrulline, ornithine, arginine, and lysine. Specific problems en countered: owing to their identical molecular weight, isoleucine and leucin e could not be quantitated separately, and, owing to their instability, glu tamine and asparagine were found to be decarboxylated to their respective a cids. Determination was linear over the concentration range tested (20 to 1 000 mu mol/L), and intraassay and interassay coefficients of variation were in the range of 10-15%. Conclusion: ESI-MSMS proved to be a highly sensiti ve, linear, and sufficiently precise method for the quantitative determinat ion of amino acids and acylcarnitines in mouse blood, allowing large-scale screening applications when speed and cost effectiveness are mandatory.