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. 2016 Aug 30;30(16):1835-45.
doi: 10.1002/rcm.7667.

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

Lili Guo  1 , Andrew J Worth  1 , Clementina Mesaros  1 , Nathaniel W Snyder  2 , Jerry D Glickson  3 , Ian A Blair  1
Affiliations

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

Lili Guo et al. Rapid Commun Mass Spectrom. .

Abstract

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.

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Figures

Figure 1
Figure 1
Scheme showing how HFIP facilitates NESI.
Figure 2
Figure 2
Work-up flow for the extraction and analysis of polar metabolites by IP-RP-UHPLC-NESI-MS for DLCL2 cells. Gradient 1 is 25 min whereas gradient 2 is 15 min in order to quickly analyze unstable redox cycling metabolites.
Figure 3
Figure 3
Representative IP-RP-UHPLC-NESI-MS chromatograms for separation improvement and sensitivity increase due to HFIP. A. Solvents used contained 5 mM DIPEA but no HFIP. B. Solvents used contained 5 mM DIPEA and 200 mM HFIP. F 1,6-bP: fructose 1,6-bisphosphate; GAP: glyceraldehyde 3-phosphate; DHAP: dihydroxyacetone phosphate; 2-PG: 2-phosphoglycerate; PEP: phosphoenolpyruvate.
Figure 4
Figure 4
Derivatization of fructose 6-phosphate and glucose-6-phosphate with phenylhydrazine.
Figure 5
Figure 5
A. Fructose 6-phosphate (F 6-P) and glucose 6-phosphate (G 6-P) without derivatization have identical retention times. B. After derivatization with phenyl-hydrazine they are chromatographically separated by more than 1 min on IP-RP-UHPLC-NESI-MS.
Figure 6
Figure 6
Quantification of isotopically labeled metabolites from DB-1 melanoma cells after derivatization with phenylhydrazine. For comparison, the carbonyl containing metabolites were quantified in parallel experiments with phenylhydrazine (PZ) derivatization (red boxes) or without derivatization (blue boxes), using gradient 1. Metabolites without a carbonyl moiety (grey boxes) were not affected by the derivatization reaction and were quantified in same IP-RP-UHPLC-NESI-MS analysis.
Figure 7
Figure 7
Levels of central energy metabolites in DLCL2 cells treated with rapamycin. The amounts of metabolites were normalized first to cell number and then to the relevant metabolites in DMSO controls. 6-PG: 6-phosphogluconate; AKG: α-ketoglutarate; OAA: oxaloacetate.
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References

    1. Grebe SK, Singh RJ. LC-MS/MS in the Clinical Laboratory - Where to From Here? Clin Biochem Rev. 2011;32:35. - PMC - PubMed
    1. King R, Bonfiglio R, Fernandez-Metzler C, Miller-Stein C, Olah T. Mechanistic investigation of ionization suppression in electrospray ionization. J. Am. Soc. Mass Spectrom. 2000;11:942. - PubMed
    1. Bonfiglio R, King RC, Olah TV, Merkle K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun. Mass Spectrom. 1999;13:1175. - PubMed
    1. Chang CH, Pearce EL. Emerging concepts of T cell metabolism as a target of immunotherapy. Nat. Immunol. 2016;17:364. - PMC - PubMed
    1. Wishart DS. Emerging applications of metabolomics in drug discovery and precision medicine. Nat. Rev. Drug Discov. 2016 - PubMed

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