Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry
Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2003-11-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520336956 |
| _version_ | 1819072310241394688 |
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| author | Bernd Lieser Gerhard Liebisch Wolfgang Drobnik Gerd Schmitz |
| author_facet | Bernd Lieser Gerhard Liebisch Wolfgang Drobnik Gerd Schmitz |
| author_sort | Bernd Lieser |
| collection | DOAJ |
| description | Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH+2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively.The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling. |
| first_indexed | 2024-12-21T17:35:41Z |
| format | Article |
| id | doaj.art-ffc13d0eec8a483e9da4519b5eea0be1 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-21T17:35:41Z |
| publishDate | 2003-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-ffc13d0eec8a483e9da4519b5eea0be12022-12-21T18:55:46ZengElsevierJournal of Lipid Research0022-22752003-11-01441122092216Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometryBernd Lieser0Gerhard Liebisch1Wolfgang Drobnik2Gerd Schmitz3Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, D-93042 Regensburg, GermanyInstitute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, D-93042 Regensburg, GermanyInstitute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, D-93042 Regensburg, GermanyInstitute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, D-93042 Regensburg, GermanySphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH+2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively.The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling.http://www.sciencedirect.com/science/article/pii/S0022227520336956high-performance liquid chromatographysphingolipidsmetabolismhigh throughput |
| spellingShingle | Bernd Lieser Gerhard Liebisch Wolfgang Drobnik Gerd Schmitz Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry Journal of Lipid Research high-performance liquid chromatography sphingolipids metabolism high throughput |
| title | Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry |
| title_full | Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry |
| title_fullStr | Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry |
| title_full_unstemmed | Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry |
| title_short | Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry |
| title_sort | quantification of sphingosine and sphinganine from crude lipid extracts by hplc electrospray ionization tandem mass spectrometry |
| topic | high-performance liquid chromatography sphingolipids metabolism high throughput |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520336956 |
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