Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma

A new analytical method has been developed and is proposed for the rapid de termination of eighteen common amino acids, including tryptophan, in plasma and dried blood spots, by liquid chromatography coupled with ionspray tand em mass spectrometry. Potentially the method can include other amino acids and can be used for the diagnosis of metabolic disease. The use of the ions pray tandem mass spectrometry approach permits extremely rapid chromatograp hic separation of all amino acids requiring less than four minutes for the analysis of each sample, after a simple sample preparation procedure. The c hromatographic separation of the analytes was achieved using a CN normal ph ase column and a water/acetonitrile/trifluoroacetic acid mobile phase at fl ow rate of 1 ml/min. The mass spectrometer was operated in multiple reactio n monitoring mode, where each analyte had its own unique precursor and prod uct ion setting. The quantitative analysis of amino acids was achieved usin g as internal standards just two representative isotopically labeled amino acids: D4-Ala and D5-Phe. Calibration is made externally by using aqueous s olutions with the same labelled amino acids as internal standards. The high specificity of tandem mass spectrometry coupled with a fast chromatographi c process is suitable for the rapid and reliable assay of metabolically sig nificant amino acids. The liquid chromatography-tandem mass spectrometry me thod is more effective than other published tandem mass spectrometry method s at distinguishing isobaric amino acids like Leu, Ile and HO-Pro and certa inly far more rapid than HPLC or ion-exchange chromatographic methods.