HILIC-Enabled 13 C Metabolomics Strategies: Comparing Quantitative Precision and Spectral Accuracy of QTOF High- and QQQ Low-Resolution Mass Spectrometry

Dynamic C-tracer-based flux analyses of in vivo reaction networks still require a continuous development of advanced quantification methods applying state-of-the-art mass spectrometry platforms. Utilizing alkaline HILIC chromatography, we adapt strategies for a systematic quantification study in non...

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Bibliographic Details
Published inMetabolites Vol. 9; no. 4
Main Authors Feith, André, Teleki, Attila, Graf, Michaela, Favilli, Lorenzo, Takors, Ralf
Format Journal Article
LanguageEnglish
Published Switzerland 02.04.2019
Subjects
LC-QQQ-MS/MS
13C-tracer studies
IDMS
Spectral accuracy
Corynebacterium glutamicum
Quantitative metabolomics
Isotopic distribution
LC-QTOF-HRMS
HILIC
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Summary:Dynamic C-tracer-based flux analyses of in vivo reaction networks still require a continuous development of advanced quantification methods applying state-of-the-art mass spectrometry platforms. Utilizing alkaline HILIC chromatography, we adapt strategies for a systematic quantification study in non- and C-labeled multicomponent endogenous extracts by LC-QTOF high resolution (HRMS) and LC-QQQ tandem mass spectrometry (MS/MS). Without prior derivatization, a representative cross-section of 17 central carbon and anabolic key intermediates were analyzed with high selectivity and sensitivity under optimized ESI-MS settings. In column detection limits for the absolute quantification range were between 6.8-304.7 (QQQ) and 28.7-881.5 fmol (QTOF) with comparable linearities (3-5 orders of magnitude) and enhanced precision using QQQ-MRM detection. Tailor-made preparations of uniformly (U) C-labeled cultivation extracts for isotope dilution mass spectrometry enabled the accurate quantification in complex sample matrices and extended linearities without effect on method parameters. Furthermore, evaluation of metabolite-specific -to- ratios (ISR ) in non-labeled extracts exhibited sufficient methodical spectral accuracies with mean deviations of 3.89 ± 3.54% (QTOF) and 4.01 ± 3.01% (QQQ). Based on the excellent HILIC performance, conformity analysis of time-resolved isotopic enrichments in C-tracer experiments revealed sufficient spectral accuracy for QQQ-SIM detection. However, only QTOF-HRMS ensures determination of the full isotopologue space in complex matrices without mass interferences.
ISSN:2218-1989
2218-1989