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An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells

The main concerns in targeted “sphingolipidomics” are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to evaluate...

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Autores principales: Morano, Camillo, Zulueta, Aida, Caretti, Anna, Roda, Gabriella, Paroni, Rita, Dei Cas, Michele
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9147510/
https://www.ncbi.nlm.nih.gov/pubmed/35629954
http://dx.doi.org/10.3390/metabo12050450
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author Morano, Camillo
Zulueta, Aida
Caretti, Anna
Roda, Gabriella
Paroni, Rita
Dei Cas, Michele
author_facet Morano, Camillo
Zulueta, Aida
Caretti, Anna
Roda, Gabriella
Paroni, Rita
Dei Cas, Michele
author_sort Morano, Camillo
collection PubMed
description The main concerns in targeted “sphingolipidomics” are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to evaluate a derivatization step for sphingoid bases quantification, to avoid interferences and improve sensitivity. We tested four protocols for the extraction of sphingolipids from human plasma, at different temperatures and durations, and two derivatization procedures for the conversion of sphingoid bases into phenylthiourea derivatives. Different columns and LC-MS/MS chromatographic conditions were also tested. The protocol that worked better for sphingolipids analysis involved a single-phase extraction in methanol/chloroform mixture (2:1, v/v) for 1 h at 38 °C, followed by a 2 h alkaline methanolysis at 38 °C, for the suppression of phospholipids signals. The derivatization of sphingoid bases promotes the sensibility of non-phosphorylated species but we proved that it is not superior to a careful choice of the appropriate column and a full-length elution gradient. Our procedure was eventually validated by analyzing plasma and erythrocyte samples of 20 volunteers. While both extraction and methanolysis are pivotal steps, our final consideration is to analyze sphingolipids and sphingoid bases under different chromatographic conditions, minding the interferences.
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spelling pubmed-91475102022-05-29 An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells Morano, Camillo Zulueta, Aida Caretti, Anna Roda, Gabriella Paroni, Rita Dei Cas, Michele Metabolites Article The main concerns in targeted “sphingolipidomics” are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to evaluate a derivatization step for sphingoid bases quantification, to avoid interferences and improve sensitivity. We tested four protocols for the extraction of sphingolipids from human plasma, at different temperatures and durations, and two derivatization procedures for the conversion of sphingoid bases into phenylthiourea derivatives. Different columns and LC-MS/MS chromatographic conditions were also tested. The protocol that worked better for sphingolipids analysis involved a single-phase extraction in methanol/chloroform mixture (2:1, v/v) for 1 h at 38 °C, followed by a 2 h alkaline methanolysis at 38 °C, for the suppression of phospholipids signals. The derivatization of sphingoid bases promotes the sensibility of non-phosphorylated species but we proved that it is not superior to a careful choice of the appropriate column and a full-length elution gradient. Our procedure was eventually validated by analyzing plasma and erythrocyte samples of 20 volunteers. While both extraction and methanolysis are pivotal steps, our final consideration is to analyze sphingolipids and sphingoid bases under different chromatographic conditions, minding the interferences. MDPI 2022-05-17 /pmc/articles/PMC9147510/ /pubmed/35629954 http://dx.doi.org/10.3390/metabo12050450 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Morano, Camillo
Zulueta, Aida
Caretti, Anna
Roda, Gabriella
Paroni, Rita
Dei Cas, Michele
An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells
title An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells
title_full An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells
title_fullStr An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells
title_full_unstemmed An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells
title_short An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells
title_sort update on sphingolipidomics: is something still missing? some considerations on the analysis of complex sphingolipids and free-sphingoid bases in plasma and red blood cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9147510/
https://www.ncbi.nlm.nih.gov/pubmed/35629954
http://dx.doi.org/10.3390/metabo12050450
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