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

Abstract

AbstractA simple procedure for the extraction and derivatization of α‐amino acids and α‐keto acids from human plasma is described. This stable isotope dilution method permits the quantification of isotopic enrichment and concentrations of several α‐keto and α‐amino acids simultaneously. The derivatization process results in methylformated, pentafluorobenzylated α‐amino acids and pentafluorobenzylated α‐keto acids. The derivatives proved to be very stable when stored at 5°C. Under electron capture chemical ionization (ECCI) conditions the derivatized amino acid produces abundant [M – PFB]− or [M – PFB – CH3OH]− anions; the α‐keto acid gives abundant [M – PFB]− anions. The method was applied to the analyses of 100 μl plasma samples in studies on protein metabolism. For the determination of concentrations [α‐ketoisocaproic acid (α‐KIC), Leu, Phe and Tyr] and enrichments ([1‐13C]‐α‐KIC, [1‐13C]‐Leu, D5‐Phe, D2‐Tyr, D4‐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.