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

Abstract

Dynamic 13C-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 13C-labeled multicomponent endogenous Corynebacterium glutamicum 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)13C-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 m+1-to-m+0 ratios (ISR1:0) 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 13C-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.

Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates

“…Targeted LC-MS measurements were performed with enhanced sensitivity on an Agilent 1200 HPLC system coupled with an Agilent 6410B triple quadrupole mass spectrometer (QQQ). Sample preparation and chromatographic separation of nucleoside phosphates by bicratic polymer-based zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) were performed as previously described (Feith et al 2019;Hildenbrand et al 2020) with following modifications: Mobile phases (constant flow rate of 0.2 ml min −1 ) were composed of aqueous buffer solutions (10 mM ammonium acetate, pH 9.2) with 90% (v/v) acetonitrile for eluent A and 10% (v/v) acetonitrile for eluent B, using the following program for gradient elution: isocratic hold 0% B for 1 min, linear gradient from 0% B to 70% B for 19 min, linear gradient from 70% B to 100% B for 2 min, isocratic hold 100% B for 2.5 min, linear gradient from 100% B to 0% B for 4 min, and equilibration to starting conditions by an isocratic hold 0% B for 12 min. Nucleoside mono-, di-, and triphosphates were detected in negative ionization mode (ESI-) with high sensitivity in selected ion monitoring (SIM) mode based on pre-optimized precursor ion transitions with a mass resolution of 0.3 u and associated MS parameters (Teleki et al 2015).…”

Comparison of l‐tyrosine containing dipeptides reveals maximum ATP availability for l‐prolyl‐l‐tyrosine in CHO cells

“…Dipeptide, amino acid, and central metabolite concentrations were determined on an HPLC system (Agilent 1200 Series) coupled with an Agilent 6410B triple quadrupole tandem mass spectrometer (QQQ-MS/MS, Agilent Technologies, Waldbronn, Germany). The LC-MS method was based on a bicratic (two-phase) zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) under alkaline mobile phase conditions without any derivatization [14,15]. Targeted metabolites were detected with high selectivity with pre-optimized precursor-toproduct ion transitions and associated MS/MS settings in multiple reaction monitoring (MRM) mode.…”

Metabolomics study of different parts of licorice from different geographical origins and their anti‐inflammatory activities

“…Metabolomics are characterized by high sensitivity, selectivity, and accuracy [15]. It has been applied to many areas, such as clinical disease diagnosis [16], botany [17], drug toxicity evaluation [18], and nutrition science [19].…”