%0 Journal Article %1 ISI:000353250900002 %A Teleki, Attila %A Sanchez-Kopper, Andres %A Takors, Ralf %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2015 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J ANALYTICAL BIOCHEMISTRY %K myown %P 4-13 %R 10.1016/j.ab.2015.01.002 %T Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry %U https://doi.org/10.1016/j.ab.2015.01.002 %V 475 %X Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90\% of the analyzed metabolites presented detection limits better than 0.5 pmol (on column), and 70\% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites. (C) 2015 Elsevier Inc. All rights reserved.

@article{ISI:000353250900002, abstract = {{Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90\% of the analyzed metabolites presented detection limits better than 0.5 pmol (on column), and 70\% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites. (C) 2015 Elsevier Inc. All rights reserved.}}, added-at = {2018-06-08T12:17:34.000+0200}, address = {{525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}}, affiliation = {{Takors, R (Reprint Author), Univ Stuttgart, Inst Biochem Engn, D-70569 Stuttgart, Germany. Teleki, Attila; Sanchez-Kopper, Andres; Takors, Ralf, Univ Stuttgart, Inst Biochem Engn, D-70569 Stuttgart, Germany.}}, author = {Teleki, Attila and Sanchez-Kopper, Andres and Takors, Ralf}, author-email = {{ralf.takors@ibvt.uni-stuttgart.de}}, biburl = {https://puma.ub.uni-stuttgart.de/bibtex/25f25938e9c5af1a039337363c1d04c9a/ralftakors}, da = {{2018-01-26}}, doc-delivery-number = {{CG4JH}}, doi = {{10.1016/j.ab.2015.01.002}}, eissn = {{1096-0309}}, funding-acknowledgement = {{Bundesministerium fur Bildung und Forschung (BMBF, Berlin, Germany) {[}0315867]; German Academic Exchange Service (DAAD, Bonn, Germany); Instituto Tecnologico de Costa Rica (ITCR, Cartago, Costa Rica)}}, funding-text = {{The authors thank Oliver Vielhauer, Olga Bungart, Benjamin Gann, Andrea Seipel, Mira Lenfers-Lucker, Lisa Junghans, and Michael Kraml (Institute of Biochemical Engineering, University of Stuttgart) for experimental support. The work was funded by the Bundesministerium fur Bildung und Forschung (Grant 0315867, BMBF, Berlin, Germany) in cooperation with Evonik Industries and was cosupported by the German Academic Exchange Service (DAAD, Bonn, Germany) and the Instituto Tecnologico de Costa Rica (ITCR, Cartago, Costa Rica).}}, interhash = {3edc3018ea64f7cd0ee45f7121348790}, intrahash = {5f25938e9c5af1a039337363c1d04c9a}, issn = {{0003-2697}}, journal = {{ANALYTICAL BIOCHEMISTRY}}, journal-iso = {{Anal. Biochem.}}, keywords = {myown}, keywords-plus = {{UNDERIVATIZED AMINO-ACIDS; PH MOBILE PHASES; ELECTROSPRAY-IONIZATION; ESCHERICHIA-COLI; QUANTITATIVE-ANALYSIS; CORYNEBACTERIUM-GLUTAMICUM; ESI-MS/MS; METABOLOMICS; EXTRACTS; MS}}, language = {{English}}, month = {{APR 15}}, number-of-cited-references = {{61}}, pages = {{4-13}}, publisher = {{ACADEMIC PRESS INC ELSEVIER SCIENCE}}, research-areas = {{Biochemistry \& Molecular Biology; Chemistry}}, times-cited = {{14}}, timestamp = {2018-06-08T10:17:34.000+0200}, title = {{Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry}}, type = {{Article}}, unique-id = {{ISI:000353250900002}}, url = {https://doi.org/10.1016/j.ab.2015.01.002}, usage-count-last-180-days = {{7}}, usage-count-since-2013 = {{46}}, volume = {{475}}, web-of-science-categories = {{Biochemical Research Methods; Biochemistry \& Molecular Biology; Chemistry, Analytical}}, year = {{2015}} }