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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: TuP-012
Session: Clinical Chemistry Applications
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

Profiling of Free Amino and Fatty Acids as a Tool for Discovery of Metabolic Disorders

Natalia V. Arkhipenko1, Marina A. Dikunets, Tim G. Sobolevsky, Svetlana A. Appolonova, Grigory M. Rodchenkov

1 Antidoping Centre, Moscow, Russian Federation

Correspondence address: Natalia V. Archipenko, Antidoping Centre, Elizavetinskiy per. 10, Moscow, 105005 Russian Federation.

Keywords: Acids, Amino; Acids, Fatty; Mass Spectrometry, Gas Chromatography; Plasma, Human.

Novel aspect: Sensitive and reproducible procedure of simultaneous GC-MS determination of fatty and amino acids in human plasma.

 

Amino acids play central role both as building blocks of proteins and as intermediates in metabolism. The identification and quantitation of amino acids in various matrices is a common task. The determination of free amino and fatty acids in biological fluids are used in clinical practice. The concentration levels of these substances are a deciding factor for diagnostics of various diseases.
To detect these compounds, various techniques are used including gas chromatography and high-resolution liquid chromatography. Although gas chromatographic methods have some advantages over HPLC, derivatization of amino and fatty acids needs to be performed before the analysis to produce volatile products.

Prior to gas chromatography, the amino and fatty acids are usually volatilized using silylation or chloroformate-induced derivatization. The latter approach seems advantageous over silylation in most applications for a number of reasons: the reaction is fast and proceeds in aqueous-organic medium; the extractant (chloroform) is volatile and nonreactive to stationary phase.
Whole blood (ca. 2-3 ml) is taken into a heparin containing tube and separated as soon as possible. Specimens are to be analysed immediately after plasma preparation. Sample pretreatment of plasma was performed by liquid-liquid extraction to remove the proteins and neutral lipids. Then the aliquots are treated with ethyl chloroformate and trifluoroethanol (under catalytic influence of pyridine) to produce the N(O,S)-ethoxycarbonyl trifluoroethyl esters of free amino acids and trifluoroethyl esters of fatty acids. After that the derivatives formed are extracted with chloroform by vigorous shaking followed by centrifugation of the solution. Organic fraction is evaporated to dryness under a stream of nitrogen. The residue is reconstituted in 50 µl of ethylacetate and 1 µl of solution is injected into a GC-MS.

Simultaneous GC-MS determination of 13 fatty and 24 amino acids in human plasma was performed directly in aqueous media using ethyl chloroformate derivatives in a single run. The sample preparation is selective and simple. The application of the selected-ion monitoring in GC-MS resulted in detection limits of 1 µg/ml plasma for all compounds. The plasma from a group of healthy donors and patients with various endocrine diseases are examined and compared.