Separation of carbohydrate isomers and anomers on poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase by hydrophilic interaction chromatography as well as determination of anomer interconversion energy barriers.

A new commercially available HPLC column, poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase (Daicel DCpak PTZ), was systematically evaluated for its carbohydrate isomer separation capability by hydrophilic interaction liquid chromatography (HILIC) with charged aerosol detection (CAD) or (tandem) mass spectrometry. Reducing sugars tend to split into two anomer peaks which makes carbohydrate isomer separations in non-derivatized form even more complicated. For practical purposes anomer separations are therefore ideally suppressed which can be accomplished by using high temperature or high pH that are both associated with fast interconversion kinetics leading to peak coalescence, or on the other hand by conditions with low chromatographic anomer selectivity. Four major hexoses (glucose, mannose, galactose, fructose), five main pentoses (ribose, ribulose, xylose, xylulose, arabinose) and five most important disaccharides (maltose, cellobiose, lactose, sucrose, trehalose) were analyzed as single carbohydrate standards by isocratic HILIC with 0.1% (v/v) formic acid and 2 mM ammonium acetate at various temperatures to study anomer interconversion equilibria in a pH-dependent manner. Rate constants of forward (α→β) and backward (β→α) anomerization and corresponding energy barriers were calculated. The energy barriers of anomerisation were in the range of around 83-91 kJ mol-1 at 298 K and the difference between forward (α→β) and backward reaction (β→α) was typically between 1-3 kJ mol-1. The systematic studies finally allowed to pick conditions for the simultaneous analysis of all 14 carbohydrates by HILIC-ESI-MS(/MS) with PTZ in gradient elution mode. A combination of carbohydrate isomer-selective LC (with PTZ), tandem MS (with carbohydrate group-selective MS1 and some species-specific SRM transitions) and a simple deconvolution strategy allowed the determination of all carbohydrates of the complex test mixture except for the disaccharide pair lactose and maltose (which can be determined as sum). Consequently, the proposed method represents a successful step towards a global glycometabolomics profiling method of mono- and disaccharides by HILIC-ESI-MS/MS.

Fu X ，Cebo M ，Ikegami T ，Lämmerhofer M ... -  《-》

Retention characteristics of poly(N-(1H-tetrazole-5-yl)-methacrylamide)-bonded stationary phase in hydrophilic interaction chromatography.

A Poly(N-(1H-tetrazole-5-yl)methacrylamide)-bonded (PTZ) stationary phase has recently gained increased attention in hydrophilic liquid chromatography (HILIC) mode for separation of polar compounds and is now commercially available as DCpak PTZ. It is chromatographically characterized in this work. The property of the new column was proven to be the most hydrophilic and acidic by separation of mixtures of purines and pyrimidines (theophylline, theobromine, uridine and 2'-deoxyuridine) in comparison to other two commercial columns Luna HILIC and LiChrospher Diol column. The retention mechanism of the new column was investigated by design-of-experiment (DoE) approach using factorial design models. Water/acetonitrile ratio in the mobile phase, buffer salt concentration and buffer pH were considered factors employing the purine/pyrimidine mixture and glucose derivatives as test samples. The resultant retention model coefficients and contour plots documented the complementary retention and selectivity profiles of the new PTZ column as compared to Diol and Luna HILIC. Moreover, it became clearly evident that the PTZ column exhibits its best HILIC performance at eluent pH ≥ 5 because the NH group in tetrazolyl moiety is dissociated under these conditions. The applicability and great potential of the new HILIC column was proven by the chromatographic separation of complex mixtures of very hydrophilic glucose and glucose derivatives (sucrose, glucosamine, glucuronic acid, glucose-1-phosphate and trehalose, glucose, maltose, glucosamine-6-phosphate, glucose-6-phosphate and gluconic acid δ-lactone) as well as monosaccharides found in N-glycans. It is concluded that the new DCpak PTZ HILIC column could have good prospects for the separation of polar compounds e.g. in metabolomics and glycomics.

Fu X ，Cebo M ，Ikegami T ，Lämmerhofer M ... -  《-》

Isomer selectivity of one- and two-dimensional approaches of mixed-mode and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry for sugar phosphates of glycolysis and pentose phosphate pathways.

In this study, the chromatographic behavior of mixed-mode and hydrophilic interaction liquid chromatography (HILIC) with the mixed-mode HILIC/strong anion-exchange (SAX) column HILICpak VT-50 2D and the two HILIC columns Atlantis Premier BEH Z-HILIC and Acquity Premier BEH Amide was assessed with regard to their separation capability of the metabolites from the glycolysis and pentose phosphate pathways. Chromatographic conditions were evaluated with the aim of achieving separation of the isomeric glycolytic phosphorylated carbohydrate metabolites free from isomeric interferences and thus allowing for selective targeted analysis by liquid chromatography with tandem mass spectrometry (MS/MS) using multiple reaction monitoring acquisition. The effects of pH values (8.0/9.0/10.0) of the ammonium bicarbonate buffer and gradient time were investigated during HILIC-MS/MS analysis, with the optimal conditions found at pH = 10.0. Separation of the pentose phosphate isomers (ribose 5- and 1-phosphate, xylulose 5-phosphate and ribulose 5-phosphate) was achieved on the mixed-mode HILIC/SAX (HILICpak VT-50 2D) column and HILIC BEH Amide column. Column performance was evaluated based on the direct comparison of chromatographic parameters, i.e. peak width at 50% and peak tailing factors of the individual metabolites. Parity plots were generated allowing a direct comparison between the normalized retention times and assessment of orthogonality of all 3 stationary phases evaluated. Separation of 7 biologically relevant hexose monophosphates metabolites turned out to be challenging by HILIC-MS/MS, with the BEH Amide providing the best individual results for such a separation. However, fructose 6-phosphate and glucose 1-phosphate co-eluted. Therefore, an on-line heart-cutting HILIC-Mixed Mode 2D-LC-QToF experiment was conducted, allowing the separation of this critical isomer pair. In this setup, the BEH Amide column in the 1D separated the majority of target metabolites, while a heart-cut of the peak from totally coeluted fructose 6-phosphate and glucose 1-phosphate was separated in the 2D with HILICpak VT50-2D column, thus allowing undisturbed determination of the glycolytic phosphorylated carbohydrate metabolites due to their chromatographic separation from hexose monophosphate metabolites. The assay specificity towards 7 common hexose monophosphates was characterized (glucose 1- and 6-phosphate, galactose 1- and 6-phosphate, fructose 6-phosphate, mannose 1- and 6-phosphate). The selectivity of some rare hexose monophosphates (allose 6-phosphate, tagatose 6-phosphate, sorbose 1-phosphate) was also tested.

Su M ，Serafimov K ，Li P ，Knappe C ，Lämmerhofer M ... -  《-》

Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed-mode hydrophilic interaction/weak anion exchange chromatography.

Hinterwirth H ，Lämmerhofer M ，Preinerstorfer B ，Gargano A ，Reischl R ，Bicker W ，Trapp O ，Brecker L ，Lindner W ... -  《-》

Simultaneous chromatographic separation of the anomers of saccharides on a polymer sulfobetaine zwitterionic stationary phase.

Yang M ，Mei H ，Jiang Y ，Zhang F ，Yu Z ，Yang B ... -  《-》