Development of an Amino Acid (in Solution) Standard Reference Material

Amino acids are the sequential components of proteins and many are essential to our physiological well-being. Their quantification by the scientific community varies in technique and application. NIST has developed a standard reference material (SRM 2389a) consisting of a mixture of 17 stable amino acids in a buffered solution for use in inter-laboratory calibrations and intra-laboratory quality control. Certified values for each amino acid were determined by a combination of isotope-dilution liquid chromatography-tandem mass spectrometry and gravimetry. Absolute quantification of amino acids in SRM 2389a is essential for characterization of routine methods and can serve as a standard for higher order measurement traceability.

Amino acid analysis is a commonly employed approach to peptide and protein characterization and quantification. A variety of methods can be used, and each has potential limitations. A standard reference material consisting of amino acids in a simple buffer can serve as a higher order reference system for temporal traceability within labs and between labs which require accurate absolute measurements of physiological biomarkers and measurands. Certification of SRM 2389a is intended to fill the need for a traceable primary standard of amino acids for the scientific community.

SRM 2389a consists of 17 amino acids in 0.1M HCl at nearly equimolar concentrations. This SRM will serve as a replacement for SRM 2389, which was certified using a combination of data from NIST and collaborating laboratories. The NIST data was based primarily upon LC-UV measurements of derivatized amino acids. Since that time, a set of isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) methods have been developed for absolute quantification of each amino acid. Value assignment of SRM 2389a represents the first time that NIST has quantified all 17 of these amino acids by isotope-dilution LC-MS/MS methodology. Certified values for SRM 2389a will be assigned as a combination of the isotope dilution results with the gravimetric data associated with the production of the SRM solution. The LC-MS/MS methodology developed for this work can also be applied to measurement of amino acids in other matrices. 

Major Accomplishments:

• A Standard Reference Material for amino acids in solution has been developed and certification measurements have been completed for all 17 amino acids.
• Isotope-dilution LC-MS/MS methodology has been implemented for quantification of amino acids.