For quantification of intracellular metabolites, mass spectrometry combined with liquid chromatography, capillary electrophoresis, or gas chromatography is currently the method of choice, especially when combined with stable isotope-labeled internal standards (SIL-ISs). Due to the difficulties in finding a biological matrix free of intracellular metabolites, a standard addition based validation is needed. Here, we present an alternative by producing a matrix with minimal signal interferences on both the analytes and their SIL-ISs. The matrix was obtained by cultivating Saccharomyces cerevisiae in [13C6]glucose/nonlabeled glucose (50:50, w/w) growth medium. The areas of both 12C6 and 13C6 fractions of ATP in the matrix were measured to be 2% of the sum of the areas of all ATP isotopes detected. The matrix allowed for spiking of both the nonlabeled and SIL-ISs and more straightforward validation. The intra- and inter-day accuracy and precision were ⩾80% and ⩽20%, respectively. The methodology was used for quantification of nucleotides, coenzymes, and redox compounds from Saccharomyces cerevisiae. The determined energy charge ratio was 0.9, whereas the Mal-CoA/Ac-CoA ratio was 0.04. The analysis of the redox compounds was challenging due to the oxidation of NADH and NADPH, when dissolved in water or tributylamine. The oxidation was reduced by dissolving them in ammonium acetate solution (pH 8.0).
- Intracellular metabolites
- Ion pair UHPLC–MS/MS
- Matrix free of the analytes
- Oxidation of NADH and NADPH
- Stable isotope-labeled internal standards