Abstract
BackgroundSugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose–phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, sugar phosphates are notoriously difficult to analyze by traditional reversed phase liquid chromatography.ResultsHere, we show a two-step derivatization of sugar phosphates by methoxylamine and propionic acid anhydride after chloroform/methanol (3:7) extraction from Populus leaf and developing wood that improves separation, identification and quantification of sugar phosphates by ultra high performance liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS). Standard curves of authentic sugar phosphates were generated for concentrations from pg to ng/μl with a correlation coefficient R2 > 0.99. The method showed high sensitivity and repeatability with relative standard deviation (RSD) < 20% based on repeated extraction, derivatization and detection. The analytical accuracy for Populus leaf extracts, determined by a two-level spiking approach of selected metabolites, was 79–107%.ConclusionThe results show the reliability of combined reversed phase liquid chromatography–tandem mass spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts.
Highlights
Sugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose–phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways
We report on the suitability of derivatizing sugar phosphates with methoxylamine and propionic acid anhydride for identification and quantification of sugar phosphates using ultra-high-performance liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS)
The analysis showed that derivatization of sugar phosphates by oximation and propionylation is an efficient method for improving the retention and separation of sugar phosphates in reversed phase (RP)-LC by making polar compounds more hydrophobic
Summary
Sugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose–phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways. Metabolomics enables comprehensive identification and quantification of small molecule metabolites that are intermediates of primary metabolic pathways, hormones and secondary metabolites in biological systems [1] Primary metabolites, such as the intermediate metabolites of central carbon metabolism, have key functions in glycolysis, the pentose–phosphate pathway. To obtain accurate and precise measurements of sugar phosphates in plant tissues, a rapid, simple and robust extraction method is required to avoid chemical degradation and quench enzymatic activities during sample preparation. Hot ethanol extraction is suitable for extracting polar and mildly non-polar metabolites [3, 5] This method is relatively simple, it requires consequtive ethanol extraction steps, during which some enzymes may remain active and cause changes in metabolite levels. In addition to these methods, trichloroacetic acid-ether extraction can be used to extract metabolites, but this method is only suitable for acid-stable and water-soluble metabolites [4]
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