Liquid chromatography method for simultaneous quantification of ATP and its degradation products compatible with both UV-Vis and mass spectrometry

Abstract

ATP and its degradation products are essential metabolic and signaling molecules. Traditionally, they have been quantified via high-performance liquid chromatography (HPLC) with UV-Vis detection while utilizing phosphate buffer mobile phase, but this approach is incompatible with modern mass detection. The goal of this study was to develop an ultra-performance liquid chromatography (UPLC) method free of phosphate buffer, to allow for analysis of adenine nucleotides with UV-Vis and mass spectrometry (MS) simultaneously. The final conditions used an Acquity HSS T3 premier column with a volatile ammonium acetate buffer to successfully separate and quantify ATP-related analytes in a standard mixture and in extracts from non-contracted and contracted mouse hindlimb muscles. Baseline resolution was achieved with all 10 metabolites, and a lower limit of quantification down to 1 pmol per inject was observed for most metabolites using UV-Vis. Therefore, this method allows for the reliable quantification of adenine nucleotides and their degradation products via UV-Vis and their confirmation and/or identification of unknown peaks via MS.

Keywords: ATP; Adenine nucleotides; Mouse; Muscle; UPLC/MS.

Figure 1.

Schematic of the adenine nucleotide degradation from ATP to uric acid. The double arrow from IMP to AMP indicates an intermediary step not shown.

Figure 2.

Composite chromatograms using A) BEH-Amide column, B) BEH-HILIC column, and C) HSS-T3 column. UA = uric acid, HX = hypoxanthine, IMP = inosine monophosphate.

Figure 3.

HSS-T3 Premier column. Mobile Phase A: 10 mM ammonium acetate (pH 4.95) in H₂O. Mobile Phase B: 75% Acetonitrile, 5% methanol, 20% H₂O with 10 mM ammonium acetate (pH 4.95). Injection volume of 5 μl. A) Full chromatogram, with 1mM standards B) Chromatogram of same column, with 100 pmol standards.

Figure 4.

A) ATP, ADP, AMP, and HX with and without citric acid at 500 pmol/inject. B) ATP, ADP, AMP, and HX with and without citric acid at 5 pmol/inject. C) a comparison of chromatograms with and without citric acid and the phosphoric acid wash.

Figure 5.

Final Chromatogram utilizing the HSS-T3 Premier column. Mobile Phase A: 10 mM ammonium acetate (pH 4.95) and 1ppm citric acid diluted in 100%. Mobile Phase B: 75% Acetonitrile, 5% methanol, 20% H₂O with 10 mM ammonium acetate (pH 4.95) and 1ppm citric acid. Post-phosphoric acid wash. Standards contain 100 pmol of each analyte and injection volume was 2μl.

Figure 6.

Representative chromatogram of non-contracted mouse tibialis anterior muscle extract at 254nm.

Figure 7.

A) Example chromatogram of extract from mouse tibialais anterior muscle at 210nm, B) Targeted MS scan for phosphocreatine (212 m/z) and creatine (132 m/z).