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The role of amino acid analysis in bioanalysis has changed from a qualitative to a quantitative technique. With the discovery of both electrospray ionization and matrix-assisted laser desorption ionization in the early 1990s, the use of amino acid analysis for qualitative analysis of proteins and peptides has been replaced by mass spectrometry. Accurate measurement of the relative molecular masses of proteins and peptides, peptide mapping, and sequencing by tandem mass spectrometry provide significantly better qualitative information than can be achieved from amino acid analysis. At NIST, amino acid analysis is used to assign concentration values to protein and peptide standard reference materials (SRMs) which, subsequently, will be used in the calibration of a wide variety of protein and peptide assays, such as those used in clinical diagnostics. It is critical that the amino acid analysis method used at NIST for SRM measurement deliver the highest accuracy and precision possible. Therefore, we have developed an amino acid analysis method that uses isotope dilution LC-MS/MS - the analytical technique routinely used at NIST to certify analyte concentrations in SRMs for a wide variety of analytes. Amino acid analysis by isotope dilution LC-MS/MS was first used to measure the concentration of bovine serum albumin in NIST SRM 927d ("bovine serum albumin, 7% solution"). We have recently refined our isotope dilution LC-MS/MS amino acid analysis method to certify the concentration of 17 amino acids in NIST SRM 2389a ("amino acids in 0.1 mol/L hydrochloric acid"). We present here our most recent method for the quantification of amino acids using isotope dilution LC-MS/MS.
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