IDENTIFICATION OF METABOLITES IN HUMAN BLOOD PLASMA BY COMPREHENSIVE TWO DIMENSIONNAL GAS CHROMATOGRAPHY-TIME-OF-FLIGHT MASS SPECTROMETRY
Gauthier Eppe, Elizabeth A. McGaw, Nathan Dodder, Bruce A. Benner Jr, Karen W. Phinney, Michele M. Schantz
Analytical Chemistry Division, Mail stop 8392, NIST Gaithersburg, MD 20899.
A new SRM Metabolites in Human Plasma (SRM1950) is being developed to provide a well-characterized reference material for metabolomics analysis platforms. The intended use for SRM 1950 is the evaluation of quantitative and qualitative analytical methods for metabolites in plasma. In this framework, we investigated the use of two dimensional gas chromatography with time-of-flight mass spectrometry (GC x GC-TOF-MS) to identify unknown polar and semi-polar low molecular weight compounds. The optimized protocol consisted of extracting the plasma with methanol according to the method developed by A et al . Prior to GC, metabolites were derivatized with silylation reagents to increase their volatility and their thermal stability. Two reagents N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide (MTBSTFA) and N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) were tested. Three column combinations Rtx-5/Rxi-17, Rtx-5/Stabilwax and Rtx-5/Rtx-Clpesticides2 were investigated in GC x GC; all provided similar separation performances. However, the non bonded Stabilwax phase used as second dimension column is limited in temperature and did not cover the complete range of derivatized metabolites. The optimized method was able to resolve approximately 300 peaks and therefore potential compounds. The identification strategy, for both reagents, was to compare experimental fragmentation mass spectra with reference spectra available in the NIST library. Additional and complementary information was obtained by the generation of retention index (RI) in the first GC dimension using of SRM 1494 containing 18 n-alkanes (C10 to C34). Several classes of metabolites were identified: amino acids, organic acids, fatty acids and sugars. Pharmaceutical residues such as analgesics were also found at trace level in this SRM.
 J. A, J. Trygg, J.Gullberg, A. I. Johansson, P. Jonsson, H. Antti, S.L. Marklund, T. Moritz, Analytical Chemistry, 77, 8086-8094, (2005)
Mentors Name: M. Schantz
Division, Laboratory: Analytical Chemistry, Organic Chemical Metrology
Room, Building Mail Stop 8392
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