Simultaneous analysis of amino and nonamino organic acids as methyl chloroformate derivatives using gas chromatography–mass spectrometry

Abstract

There are numerous amino and nonamino organic acids that are intermediates in cellular metabolism. In the study of metabolic pathways, information about the concentration of these metabolites inside the cells is of crucial interest. Furthermore, many microorganisms overproduce a variety of amino and organic acids and secrete them to the extracellular medium, and this is explored for the production of many different chemicals in the biotechnology industry [1,2]. Several chromatographic methods, including liquid chromatography (LC), gas chromatography (GC), and gas chromatography–mass spectrometry (GC-MS), have been employed to analyze metabolites levels in biological samples [3–5, and others]. Few methods, however, enable simultaneous analysis of amino and nonamino organic acids in a single run [6,7], and to our knowledge none of these achieves analysis of a large number of diand tricarboxylic acids such as intermediates from the trichloroacetic acid cycle. GC-MS technology allows simultaneous detection of different classes of metabolites within a single analysis [8]. The determination of amino and nonamino organic acids by GC-MS methods requires the conversion of these compounds to volatile derivatives. The most popular derivatization approach is based on silylation of organic compounds, which are converted to their alkyl–silyl derivatives (i.e., by reacting with hexamethyldisilazane and obtaining trimethylsilyl derivatives) [3,7,9,10, and others]. Some disadvantages with this derivatization technique are silylation under anhydrous conditions, heating of the sample for a given period,