Critical considerations for trimethylsilyl derivatives of 24 primary metabolites measured by gas chromatography–tandem mass spectrometry

Abstract

Cellular reactions are very important for biological systems and understanding metabolic changes is therefore crucial. Here, a gas chromatography–tandem mass spectrometry method based on multiple reaction monitoring is presented, increasing specificity and sensitivity of the method. Twenty-four metabolites of the primary carbon metabolism were selected for method development as representatives of different analyte classes (carbohydrates and their phosphates, amino acids, organic acids). The derivatization strategy necessary for analysis includes trimethylsilyl derivatization or its combination with methoximation. Derivatization products and reaction kinetics were carefully studied and compared. It is shown in detail that mere silylation results in up to seven monosaccharide derivatives and that irregular derivatization products were observed for particular amino acids showing either one or two silylation products for the ε-amino groups. Additionally, it was found that there is not a defined endpoint for this reaction. Methoximation/silylation was also optimized and studied in detail (time, temperature, kinetics) and showed reduced complexity for the derivatization products. However, some metabolites exhibited significantly lower signal responses. Most interestingly, it was observed that in the presence of amino acids the derivatization products for monosaccharides are altered when compared to monosaccharide standards, as demonstrated by the detailed discussion of glucose derivatization in the presence of lysine.