Determination of isotopic ratios of L-leucine and L-phenylalanine and their stable isotope labeled analogues in biological samples by gas chromatography/triple-stage quadrupole mass spectrometry.

Abstract

A gas chromatographic/triple-stage quadrupole mass spectrometric (GC/MS/MS) method for measuring very low levels of enrichment of [5,5,5-2H3]-L-leucine and [ring-13C6]-L-phenylalanine in plasma and lipoprotein hydrolysates is described. The amino acids were derivatized to their N-heptafluorobutyryl isobutyl ester derivatives and the isotope ratio was determined by GC/MS/MS in the negative-ion chemical ionization mode. Parent ions were the [M-HF]- ions and fragment ions used for quantification were [P-2HF-C3H7]- (leucine) and [P-HF-OC4H9]- (phenylalanine), respectively. The limit of quantification was about 10 pg of the labeled compound co-eluting with 20 ng of the endogenous compound. The calibration curves were linear in the investigated range from 0.1% to 100% of the labeled compound. In biological samples, the higher selectivity of GC/MS/MS compared with GC/MS was demonstrated.