Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed. To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions. We present a double-labelling LC–MS approach with a 13C-labeled yeast cellular extract as complex surrogate matrix, and 13C15N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates. While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions. Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.