Diisopropylethylamine/hexafluoroisopropanol-mediated ion-pairing ultra-high-performance liquid chromatography/mass spectrometry for phosphate and carboxylate metabolite analysis: utility for studying cellular metabolism

RATIONALE: Mass spectrometric (MS) analysis of low molecular weight polar metabolites can be challenging because of poor chromatographic resolution of isomers and insufficient ionization efficiency. These metabolites include intermediates in key metabolic pathways, such as glycolysis, the pentose phosphate pathway, and the Krebs cycle. Therefore, sensitive, specific, and comprehensive quantitative analysis of these metabolites in biological fluids or cell culture models can provide insight into multiple disease states where perturbed metabolism plays a role.METHODS: An ion-pairing reversed-phase ultra-high-performance liquid chromatography (IP-RP-UHPLC)/MS approach to separate and analyze biochemically relevant phosphate- and carboxylic acid-containing metabolites was developed. Diisopropylethylamine (DIPEA) was used as an IP reagent in combination with reversed-phase liquid chromatography (RP-LC) and a triple quadrupole mass spectrometer using selected reaction monitoring (SRM) and negative electrospray ionization (NESI). An additional reagent, hexafluoroisopropanol (HFIP), which has been previously used to improve sensitivity of nucleotide analysis by UHPLC/MS, was used to enhance sensitivity.RESULTS: HFIP versus acetic acid, when added with the IP base, increased the sensitivity of IP-RP-UHPLC/NESI-MS up to 10-fold for certain analytes including fructose-1,6-bisphosphate, phosphoenolpyruvate, and 6-phosphogluconate. It also improved the retention of the metabolites on a C18 reversed-phase column, and allowed the chromatographic separation of important isomeric metabolites. This methodology was amenable to quantification of key metabolites in cell culture experiments. The applicability of the method was demonstrated by monitoring the metabolic adaptations resulting from rapamycin treatment of DB-1 human melanoma cells.CONCLUSIONS: A rapid, sensitive, and specific IP-RP-UHPLC/NESI-MS method was used to quantify metabolites from several biochemical pathways. IP with DIPEA and HFIP increased the sensitivity and improved chromatographic separation when used with reversed-phase UHPLC.