Comparing predefined derivatisation parameters for GC-MS analysis of selected organic acids
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Organic acids are intermediates of numerous biochemical pathways in living organisms. They can be detected in biological fluids which make it possible to obtain a representative profile of the functioning of biochemical pathways. General analysis of these acids is based on their distinguishable chemical properties. Chemically, organic acids refer to water-soluble compounds with one or more carboxylic group and often consist of other functional groups. The analysis of organic acids is commonly used for the diagnosis of inborn errors of metabolism, by using gas chromatography-mass spectrometry (GC-MS), which can be seen as the gold standard method. For GC-MS analysis of organic acids, derivatisation prior to analysis is necessary to improve the volatility, separation, selectivity and thermal stability of the compounds. Silylation is a common derivatisation method for organic acids, however disadvantages such as long incubation periods at high temperatures, lack of repeatability and the formation of multiple derivatives that result in multiple peaks on a chromatogram often occur. Therefore, research into the optimum silylation reaction conditions for organic acids are needed. The aim of this investigation was to compare the outcome of predefined silylation derivatisation parameters required for thermal- and microwave-assisted derivatisation, as a prerequisite for GC-MS analysis of selected organic acids. The organic acids were selected through the consideration of their functional groups and physiological concentration to ensure a representative group of the broad organic acid class. Guided by literature, the silylation conditions included: temperatures (50, 60, 70 and 85 °C) in combination with reaction times (30, 60, 90 and 120 min) and microwave energies (150, 230, 350 and 450 W) in combination with reaction times (1.5, 2.0, 3.0 and 4.0 min). Organic acid standards were derivatised and analysed using GC-MS in single ion monitoring mode. Data were processed, and statistical comparisons were performed. Coefficient of variance (CV) was used as the main performance criterion in this study and used as a mean to compare the results. Between conventional thermal- and microwave-assisted derivatisation, conventional thermal derivatisation was found to provide lower variation and to be more robust. Further it was found that the use of methoxymation was beneficial for repeatability of some organic acids, making adapted thermal derivatisation the preferred type. Individual organic acids performed differently at temperature, microwave energy and reaction time increments with inconsistent pattern towards higher/lower temperature or microwave energy and longer/shorter reaction time. Derivatisation efficiency was found to be largely influenced by the structure of the compound, with better repeatability when derivatising only carboxyl groups. From all conditions investigated, 60°C for 30 min was identified as the condition within adapted thermal derivatisation to provide the least variation for the included organic acids. For thermal derivatisation the condition at 70°C for 90 min gave the lowest CV values. Microwave-assisted derivatisation resulted in the lowest CV values at 150 W for 1.5 min and for adapted microwave-assisted derivatisation, this was true at 350 W for 3.0 min. This study demonstrates that derivatisation of organic acids should be done with care as the derivatisation parameter intervals (or the exactness thereof) largely influences the repeatability.