QUANTIFICATION OF AMINO ACIDS IN HUMAN PLASMA USING COMPREHENSIVE TWO DIMENSIONAL GAS CHROMATOGRAPHY TIME OF FLIGHT MASS SPECTROMETRY
Elizabeth A. McGaw, Mark S. Lowenthal, and Karen W. Phinney
Analytical Chemistry Division, CSTL, NIST
Metabolomics is an emerging field focused on analysis of low-molecular-weight compounds in complex biomatrices. These approaches are qualitative, quantitative, or a combination and can offer insights into mechanisms of disease or markers for diagnostics. NIST is developing a Standard Reference Material, Metabolites in Human Plasma (SRM 1950), to serve as a common material for researchers to evaluate new platforms in this field. Both qualitative and quantitative information will be provided to the users of this material. Free amino acids have been identified as a class of metabolites to be quantified in the SRM and this will be the first serum- or plasma-based SRM characterized for amino acids.
Two distinct derivatizing agents (N-Methyl-N-[tert-butyldimethyl-silyl]trifluoroacetimide, MTBSTFA and propyl chloroformate, PCF) were used to quantify the amino acids with gas chromatography time of flight mass spectrometry (GC-TOFMS) and two dimensional gas chromatography time of flight mass spectrometry (GCxGC-TOFMS). There are advantages and disadvantages to each derivatizing agent. The MTBSTFA derivatives are more broadly applicable allowing multiple classes of metabolites to be analyzed simultaneously. However, in GC and to some degree in GCxGC these additional components, particularly glucose because of its high concentration, can cause interferences that complicate quantitation. The advantage of the GCxGC technique is the higher resolution and sensitivity which may overcome the problem of interfering components. The PCF derivatization is more selective for amino acids which removes the major interfering peaks. This derivatization can be performed in aqueous media and is relatively quick compared to silylation. There are some amino acids such as glutamic acid and pyroglutamic acid that co-elute and have the same mass fragment so they are unable to be quantitated with PCF derivatization.
Eighteen amino acids in SRM 1950 were accurately and precisely quantified by isotope-dilution using GC-TOFMS with both MTBSTFA and PCF and using GCxGC-TOFMS with MTBSTFA, as well as with an orthogonal technique, liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS). The results are very comparable between the orthogonal techniques giving high confidence in the results and demonstrating that different techniques provide similar analytical results.