Highly selective methods for the analysis of intermediate metabolites involved in glycolysis and phosphate pentose pathways are essential for metabolism and metabolic flux studies. However, the successful separation of phosphorylated compounds is difficult due to their high polarity, as well as their structural isomers. In this study, phosphorylated compounds in spiked serum samples were analyzed using capillary electrophoresis tandem mass spectrometry (CE-MS/MS) and gas chromatography (GC)-MS/MS. Following liquid-liquid extraction, ultrafiltration and derivatization steps were needed to perform CE-MS/MS and GC-MS/MS, respectively. The CE-MS/MS method allowed for the identification and quantification of all 15 biologically important phosphorylated compounds, whereas only 13 compounds were identified and quantified by GC-MS/MS. Both methods demonstrated wide linear ranges, good interday (<9.6%: CE-MS/MS; <14.7%: GC-MS/MS) and intraday (<13.0%: CE-MS/MS; <14.9%: GC-MS/MS) variability, and limits of detection (LODs) in the ranges of 0.25-2 and 0.05-0.5 μmol/L for CE-MS/MS and GC-MS/MS, respectively. In the phosphorylated compound stability test, the instability of glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) was observed during freeze-thaw and long-term storage due to reversible isomerization. The results of CE-MS/MS and GC-MS/MS analysis showed that the concentrations of phosphorylated compounds determined using the two methods matched closely, while that of glycerol 3-phosphate (G3P) showed some variability in cell extracts. Therefore, while both CE-MS/MS and GC-MS/MS are suitable for analyzing metabolites resulting from the glycolysis and pentose phosphate pathways, additional validation is needed for some compounds, depending on the background matrix.
Keywords: CE–MS/MS; Cell extract; GC–MS/MS; Human serum; Intermediates of glycolysis and pentose phosphate pathways; Method validation.
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