Journal
TALANTA
Volume 205, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2019.120161
Keywords
Nucleotide; HPLC-MS; HILIC; ppGpp; Stringent response; Escherichia coli
Categories
Funding
- Czech ministry of health [17-29680A]
- Czech Science Foundation [15-11711S]
- Czech ministry of Education and Sport via JPIAMR [8F19006]
- Swedish Research council [2017-03783]
- Charles University, Department of Analytical Chemistry - Specific University Research [SVV260440]
- EU via JPIAMR [8F19006]
- Swedish Research Council [2017-03783] Funding Source: Swedish Research Council
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Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach for determination of the intracellular concentrations of > 25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.
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