Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry Rende, Umut; Niittylae, Totte; Moritz, Thomas Published in: Plant Methods DOI: 10.1186/s13007-019-0514-9 Publication date: 2019 Document version Publisher's PDF, also known as Version of record Document license: CC BY Citation for published version (APA): Rende, U., Niittylae, T., & Moritz, T. (2019). Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry. Plant Methods, 15, [127]. https://doi.org/10.1186/s13007-019-0514-9 Download date: 26. Sep. 2021 Rende et al. Plant Methods (2019) 15:127 https://doi.org/10.1186/s13007-019-0514-9 Plant Methods METHODOLOGY Open Access Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry Umut Rende1, Totte Niittylä1 and Thomas Moritz1,2* Abstract Background: 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. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, sugar phosphates are notoriously difcult to analyze by traditional reversed phase liquid chromatography. Results: Here, we show a two-step derivatization of sugar phosphates by methoxylamine and propionic acid anhy- dride after chloroform/methanol (3:7) extraction from Populus leaf and developing wood that improves separation, identifcation and quantifcation 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 coefcient 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%. Conclusion: The results show the reliability of combined reversed phase liquid chromatography–tandem mass spec- trometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts. Keywords: Sugar phosphates, UHPLC–ESI–MS, Q-TOF, QqQ-MS, Extraction, Two-step derivatization Background and tricarboxylic acid (TCA) cycle. Tese metabolites Metabolomics enables comprehensive identifcation are classifed as sugar phosphates, nucleotides, nucleo- and quantifcation of small molecule metabolites that tide sugars, carboxylic acids and phospho-carboxylic are intermediates of primary metabolic pathways, hor- acids. Sugar phosphates act as important intermediates mones and secondary metabolites in biological sys- in energy metabolism and provide the starting point for tems [1]. Primary metabolites, such as the intermediate most biosynthetic processes in prokaryotic and eukary- metabolites of central carbon metabolism, have key otic cells. In plants, the hexose-phosphate pool, consist- functions in glycolysis, the pentose–phosphate pathway ing of interconvertible six carbon sugars glucose-6-P, glucose-1-P and fructose-6-P [2], provides carbon for cell wall and starch biosynthesis in addition to glycolysis and *Correspondence: [email protected] the pentose phosphate pathway. Hence, comprehensive 1 Department of Forest Genetics and Plant Physiology, Umeå Plant analysis of plant primary metabolism requires a robust Science Centre, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden method for quantifying sugar phosphates. Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rende et al. Plant Methods (2019) 15:127 Page 2 of 10 Sugar phosphates are chemically unstable and sensitive in the column [10–13]. However, it has been reported to enzymatic degradation during extraction. Terefore, that IPC is not suitable for separation of isomer sugar to obtain accurate and precise measurements of sugar phosphates, and removal of ion-pairing reagents phosphates in plant tissues, a rapid, simple and robust from the chromatographic system is often problem- extraction method is required to avoid chemical degra- atic as their residues may afect MS analysis. Another dation and quench enzymatic activities during sample approach is to combine anion chromatography (AIC) preparation. Depending on the metabolite of interest, the with mass spectrometry. With this approach, it is possi- most common way of avoiding chemical and enzymatic ble to separate most of the metabolites involved in glyc- metabolite degradation during extraction is to apply olysis and the TCA cycle with good reproducibility and low or high temperatures to homogenized plant tissues sensitivity [14]. However, one drawback, besides need- in organic solvents or a mixture of solvents [3–6]. For ing a separate ion chromatograph, is the complexity of example, chloroform–methanol–water extraction at low the instrumentation, requiring membrane devices for temperature is suitable for extracting water-soluble and proton-potassium exchange and a carbonate remover. organic-soluble metabolites [6, 7]. In contrast, hot etha- Terefore, the AIC system requires a highly trained nol extraction is suitable for extracting polar and mildly operator. In contrast to reversed phase (RP)-LC, hydro- non-polar metabolites [3, 5]. However, although this philic interaction liquid chromatography (HILIC) sepa- method is relatively simple, it requires consequtive eth- rates polar compounds according to their hydrophilic anol extraction steps, during which some enzymes may interaction with the stationary phase [15]. For HILIC remain active and cause changes in metabolite levels. analysis, the sample solution should contain more In addition to these methods, trichloroacetic acid-ether than 50% organic solvent (mostly acetonitrile), which extraction can be used to extract metabolites, but this decreases the solubility of polar compounds. Tis limits method is only suitable for acid-stable and water-soluble usage of aqueous solutions, in which sugar phosphates metabolites [4]. readily dissolve [16, 17]. Although in HILIC analy- Analysis of sugar phosphates is usually performed by sis it is difcult to separate isomeric sugar phosphates mass spectrometry (MS) connected to capillary electro- and it has been shown that for some metabolites, the phoresis (CE), gas chromatography (GC) or liquid chro- reproducibility of HILIC is poor [18], there are reports matography (LC). CE–MS has high separation efciency where diferent sugar phosphates have been analyzed and sensitivity for measuring sugar phosphates, but it by HILIC chromatography [19, 20]. requires very low injection volumes (nL) and thereby RP-LC is not suitable for highly polar compounds fails to detect sugar phosphates at low concentrations [8]. because they are not retained on a C18 column. To GC–MS analysis is suitable for identifying and quantify- overcome this problem, diferent types of derivatiza- ing volatile compounds. Hence, nonvolatile compounds, tion techniques have been used to improve retention in such as neutral sugars and sugar phosphates, must be LC–MS. For example, 2-aminopyridine [21], p-amino derivatized prior to GC–MS analysis, e.g. by using a benzoic ethyl ester [22] or 4-(3-methyl-5-oxo-2-pyra- combination of oximation and trimethylsilylation deri- zolin-1-yl) benzoic acid [23] reagents have been used vatization [7]. Recently, a pentafuorobenzyl oxime and in diferent studies to improve chromatographic reten- trimethylsilyl derivatization strategy combined with tion of highly polar compounds. However, these deri- GC-negative chemical ionization MS showed promising vatization methods are tedious and not reliable due to results for detecting sugar phosphates in cell cultures [9]. incomplete derivatization. It has also been shown that Among combined chromatography–mass spectrometry derivatization of highly polar compounds by either techniques, LC–MS is commonly used for polar com- propionylation or benzoylation can improve chromato- pounds and is compatible with aqueous phases obtained graphic retention in LC–ESI–MS [24]. during the extraction. In this study, we report on the suitability of derivat- Diferent LC–MS techniques have been developed to izing sugar phosphates with methoxylamine and propi- analyze a variety of metabolites, including sugar phos- onic acid anhydride for identifcation and quantifcation phates. For instance, ion pair chromatography (IPC), of sugar phosphates using ultra-high-performance liq- which is a type of reversed phase LC with a hydro- uid chromatography–electrospray ionization–mass phobic stationary phase, can be used to separate ionic