Analysis of Selected Sugars and Sugar Phosphates in Mouse Heart Tissue by Reductive Amination and Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Abstract

Sensitive and reliable analysis of sugars and sugar phosphates in tissues and cells is essential for many biological and cell engineering studies. However, the successful analysis of these endogenous compounds in biological samples by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is often difficult because of their poor chromatographic retention properties in reversed-phase LC, the complex biological matrices, and the ionization suppression in ESI. This situation is further complicated by the existence of their multiple structural isomers in vivo. This work describes the combination of reductive amination using 3-amino-9-ethylcarbazole, with a new LC approach using a pentafluorophenyl core–shell ultrahigh performance (UP) LC column and methylphosphonic acid as an efficient tail-sweeping reagent for improved chromatographic separation. This new method was used for selected detection and accurate quantitation of the major free and phosphorylated reducing sugars in mouse heart tissue. Among the detected compounds, accurate quantitation of glyceraldehyde, ribose, glucose, glycerylaldehyde-3-phosphate, ribose-5-phosphate, glucose-6-phosphate, and mannose-6-phosphate was achieved by UPLC/multiple-reaction monitoring (MRM)-MS, with analytical accuracies ranging from 87.4% to 109.4% and CVs of ≤8.5% (n = 6). To demonstrate isotope-resolved metabolic profiling, we used UPLC/quadrupole time-of-flight (QTOF)-MS to analyze the isotope distribution patterns of C3 to C6 free and phosphorylated reducing sugars in heart tissues from 13C-labeled wild type and knockout mice. In conclusion, the preanalytical derivatization-LC/ESI-MS method has resulted in selective determination of free and phosphorylated reducing sugars without the interferences from their nonreducing structural isomers in mouse heart tissue, with analytical sensitivities in the femtomole to low picomole range.

Highly efficient solid-phase derivatization of sugar phosphates with titanium-immobilized hydrophilic polydopamine-coated silica

“…As a very low abundant sugar phosphate, E4P often couldn't be quantified in the similar cell samples owing to the insufficient sensitivity of the previous methods [6,12]. The developed method in this work could greatly improve the sensitivity of E4P by three orders of magnitude compared with that of the previous liquid-phase derivatization [12], and thus E4P could be well quantified even from only thousands of cells. Fig.…”

“…As a comparison, the sugar phosphates were derivatized by a liquid-phase approach on the basis of the previously reported method [12]. The relative recovery (Rec) was calculated by the following formula:…”

“…Among them, LC-MS has become the most widely used technique for its high sensitivity and resolution [12]. However, the determination of sugar phosphates is still a challengeable task because of their low endogenous concentrations, poor chromatographic retention on reversed-phase liquid chromatography (RPLC) as well as the ionization suppression from complex matrix interference of biological samples.…”

“…Ion pairing reagents, like tributylamine were often employed as the counterion to improve chromatographic retention on RPLC [7,9] or hydrophilic interaction liquid chromatography (HILIC) was employed for determination of the sugar phosphates [13,14], However, the extra ionization suppression resulting from the ion pairing reagents in RPLC or the volatile salts in HILIC could seriously reduce the detection sensitivity of sugar phosphates [12].…”

“…Meanwhile, the derivatization approach based on reductive amination has been employed for the LC-MS analysis of sugar phosphates in biological samples by using aniline or 3-amino-9-ethylcarbazole (AEC) as derivative reagents [12,19]. However, the derivatization was often carried out in the liquid phase, which had inherent disadvantages including time-consuming, residual derivative reagent and low efficiency of derivatization.…”