Simultaneous determination of concentrations and stable isotope enrichments of α-ketoisocaproic acid, leucine, phenylalanine and tyrosine in blood plasma by gas chromatography/negative ion mass spectrometry

A simple procedure for the extraction and derivatization of alpha-amino acids and alpha-keto acids from human plasma is described. This stable isotope dilution method permits the quantification of isotopic enrichment Pod concentrations of several alpha-keto and alpha-amino acids simultaneously. The derivatization process results in methylformated, pentafluorobenzylated alpha-amino acids and pentafluorobenzylated alpha-keto acids. The derivatives proved to be very stable when stored at 5 degrees C. Under electron capture chemical ionization (ECCI) conditions the derivatized amino acid produces abundant [M-PFB](-) or [M-PFB-CH3OH](-) anions; the alpha-keto acid gives abundant [M-PFB](-) anions. The method was applied to the analyses of 100 mu l plasma samples in studies on protein metabolism. For the determination of concentrations [alpha-ketoisocaproic acid (alpha KIC), Leu, Phe and Tyr] and enrichments ([1-C-13]-alpha-KIC, [1-C-13]-Leu, D-5-Phe, D-2-Tyr, D-4-Tyr), resulting from administered tracers, the corresponding [M-PFB](-) or [M-PFB-CH3OH](-) anions were monitored. Higher labelled analogues were used as internal standards. The overall sensitivity was sufficient to study isotopic enrichments in the range 0.2-10 at. % excess.