Simultaneous quantification of plasma levels of α-ketoisocaproate and leucine by gas chromatography-mass spectrometry

This chapter describes a method implying electron ionization–gas chromatography–mass spectrometry (EI-GC-MS) measurement of tert-butyldimethylsilyl (TBDMS) and methyloxime tert-butyldimethylsilyl derivatives of leucine and α-ketoisocaproic acid (KIC), respectively. Plasma levels of the two compounds are determined accurately with norleucine and α-ketovaleric acid (KVA) as internal standards. [1-13C]Leucine and [1-13C]KIC enrichment can also be evaluated by the present method in plasma samples treated with the labeled 13C isotopomers. Both sample preparation and GC–MS analysis are simple and rapid, so that several samples can be analyzed daily. Under selected conditions silylation with N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide allows derivatization of both leucine and KIC and is efficient enough for the evaluation of plasma levels of the two compounds in the EI ionization mode. Compared with trimethylsilyl derivatives, TBDMS derivatives of amino acids offer the advantage of much higher stability to hydrolysis, improved separation by GC, and an intense molecular weight-indicative peak, [M-57]+. Good linearity and high sensitivity are obtained. Optimal linearity obtained for [1-13C]leucine–leucine calibration curves suggests that [1-13C]leucine may be used instead of norleucine as the internal standard.