Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry

Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis

“…Purine nucleotide profiling is an analytical challenge because of their high polarity, instability, and chemical similarities under traditional reverse phase methodologies (17). Here we applied hydrophilic interaction liquid chromatography-based ultra high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) to specifically quantify each individual cellular purine nucleotide in purinosome-rich HeLa cells (cultured in PϪ medium) and in normal HeLa cells (cultured in non-dialyzed FBS-based purine-rich (Pϩ) medium), including IMP, the product of the de novo pathway, and two direct products of subsequent branch pathways, AMP and GMP, as well as their corresponding phosphorylated species: ADP, ATP, GDP, and GTP.…”

Analytical Approaches for the Quantitation of Redox-active Pyridine Dinucleotides in Biological Matrices

“…For Chinese hamster ovary (CHO) cell culture different extraction solvents were tested [8]. Acetonitrile, ethanol, methanol, acetonitrile: water (8:2), ethanol: water (8:2), methanol: water (8:2), 0.1 M formic acid, 0.5 M perchloric acid (PCA) and 0.1 M formic acid in methanol were used.…”

“…When using IP reagents, the stationary phase is a usual reversed phase column. Ion-pair reagents are normally alkylamines, such as dibutylammonium acetate (DBAA) [32], tributylamine (TBA) [5-7, 29, 31], dimethylhexylamine (DMHA) [8,34], dibutylamine (DBA) [4], hexylamine (HA) [7] for negative ionization mode. As nucleotides are detected in negative mode, ion suppression is less problematic.…”

“…The concentration can be reduced if the alkyl chain of the used IP is longer as it helps interaction between IP reagent and the apolar stationary phase. The effect of different concentrations of DMHA was tested by Cordell et al [8]. They varied the concentration of the ion-pair agent between 0.5 mM and 5 mM.…”

“…The most suitable mobile phases usually consist of methanol or acetonitrile and water with a buffer of volatile salts (i.e. ammonium-acetate, ammonium formate) and in some cases, volatile ion-pair reagents, among others dibutylamine (DBA) [4], tributylamine (TBA) [5][6][7], dimethylhexylamine (DMHA) [8]. High resolution is fundamental in untargeted metabolomics, but in targeted metabolic profiling, lower resolution tandem mass spectrometers, such as triple quadrupoles also provide the information needed [9].…”