Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry
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 simpl...
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| Format: | Article |
| Language: | English |
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BMC
2019-11-01
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| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-019-0514-9 |
| _version_ | 1818340896845856768 |
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| author | Umut Rende Totte Niittylä Thomas Moritz |
| author_facet | Umut Rende Totte Niittylä Thomas Moritz |
| author_sort | Umut Rende |
| collection | DOAJ |
| description | 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 difficult to analyze by traditional reversed phase liquid chromatography. Results Here, 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 R 2 > 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 spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts. |
| first_indexed | 2024-12-13T15:50:11Z |
| format | Article |
| id | doaj.art-4c104083445e418881a72b29c8f88838 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-13T15:50:11Z |
| publishDate | 2019-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-4c104083445e418881a72b29c8f888382022-12-21T23:39:31ZengBMCPlant Methods1746-48112019-11-0115111010.1186/s13007-019-0514-9Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometryUmut Rende0Totte Niittylä1Thomas Moritz2Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural SciencesDepartment of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural SciencesDepartment of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural SciencesAbstract 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 difficult to analyze by traditional reversed phase liquid chromatography. Results Here, 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 R 2 > 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 spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts.http://link.springer.com/article/10.1186/s13007-019-0514-9Sugar phosphatesUHPLC–ESI–MSQ-TOFQqQ-MSExtractionTwo-step derivatization |
| spellingShingle | Umut Rende Totte Niittylä Thomas Moritz Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry Plant Methods Sugar phosphates UHPLC–ESI–MS Q-TOF QqQ-MS Extraction Two-step derivatization |
| title | Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry |
| title_full | Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry |
| title_fullStr | Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry |
| title_full_unstemmed | Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry |
| title_short | Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry |
| title_sort | two step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography tandem mass spectrometry |
| topic | Sugar phosphates UHPLC–ESI–MS Q-TOF QqQ-MS Extraction Two-step derivatization |
| url | http://link.springer.com/article/10.1186/s13007-019-0514-9 |
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