Stepwise extraction, chemical modification, GC–MS separation, and determination of amino acids in human plasma#

Abstract

AbstractA method for isolation of enriched fractions of amino acids from human plasma followed by derivatization, gas chromatography separation and mass spectrometry identification is described. The method involves a stepwise extraction of plasma constituents with the use of two solvents: (a) extraction with methanol yields a concentrate of polyols, urea, carbohydrates and most long‐chain saturated and unsaturated aliphatic acids, and (b) further solubilization with water produces mainly a concentrate of amino acids. Chemical modification of amino acids with methyl chloroformate/methanol gives rise to methyl esters of methoxycarbonyl derivatives. The derivatization products are stable and quite suitable for gas chromatography with mass spectrometry analysis. The electron ionization mass spectra of the derivatization products reveal specific fragmentation patterns applicable for structure elucidation. The efficiency of the method is demonstrated by gas chromatography with mass spectrometry identification of 19 amino acids as their methyl esters of methoxycarbonyl derivatives in Standard Reference Material 1950 Metabolites in Frozen Human Plasma.