Isotopic Determination of Organic Keto Acid Pentafluorobenzyl Esters in Biological Fluids by Negative Chemical Ionization Gas Chromatography/Mass Spectrometry

A rapid, single-step procedure (or the extraction and derivatization of organic α-keto acids from microliter quantities of human plasma has been developed. The keto acids were analyzed as the pentafluorobenzyl (PFB) ester by methane negative chemical ionization gas chromatography/mass spectrometry. The PFB esters possess excellent chromatographic properties and required no further derivatization to block the keto group. They fragment to produce intense carboxylate anions, often as the sole ion in the spectrum, and offer detection limits below 1 pmol. This derivative is suitable for isotopic analysis of organic keto adds because R does not Introduce any additional isotopic complexity Into the target molecule. Normal human plasma 4-methyl-2-oxopentanolc acid levels were 34.9 ± 5.3 μmol^.L.⁻¹ and could be determined with 1.1% precision by Isotope dilution GC/MS. We have used this procedure to study leucine and 4-methyl-2-oxopentanolc acid metabolism by using stable isotopically labeled tracers in a variety of normal and abnormal conditions.