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Quantitative analysis of intracellular nucleoside triphosphates and other polar metabolites using ion pair reversed-phase liquid chromatography coupled with tandem mass spectrometry

Wu, Jianmei, Zhang, Yingtao, Wiegand, Richard, Wang, Jian, Bepler, Gerold, Li, Jing
Journal of Chromatography B 2015 v.1006 pp. 167-178
acetates, adenosine triphosphate, electrospray ionization mass spectrometry, guanosine triphosphate, hydrophilicity, metabolites, nitroprusside, nucleosides, pH, quantitative analysis, reversed-phase liquid chromatography, tandem mass spectrometry, triethylamine, uridine triphosphate
Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA)—hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100mM, pH 7.0), the combination of HFIP (100mM) and TEA (8.6mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5nM for CTP, UTP, dATP, dCTP, and dTTP, at 1nM for ATP, and at 5nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (<15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.