SIMULTANEOUS DETERMINATION OF CONCENTRATIONS AND STABLE-ISOTOPE ENRICHMENTS OF ALPHA-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-amin o acids and alpha-keto acids from human plasma is described. This stab le isotope dilution method permits the quantification of isotopic enri chment Pod concentrations of several alpha-keto and alpha-amino acids simultaneously. The derivatization process results in methylformated, pentafluorobenzylated alpha-amino acids and pentafluorobenzylated alph a-keto acids. The derivatives proved to be very stable when stored at 5 degrees C. Under electron capture chemical ionization (ECCI) conditi ons 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 concentration s [alpha-ketoisocaproic acid (alpha KIC), Leu, Phe and Tyr] and enrich ments ([1-C-13]-alpha-KIC, [1-C-13]-Leu, D-5-Phe, D-2-Tyr, D-4-Tyr), r esulting from administered tracers, the corresponding [M-PFB](-) or [M -PFB-CH3OH](-) anions were monitored. Higher labelled analogues were u sed as internal standards. The overall sensitivity was sufficient to s tudy isotopic enrichments in the range 0.2-10 at. % excess.