Abstract
A unique monophasic extraction system coupled with LC/MS/MS to reduce matrix effects for sphingolipid analysis was developed. A solvent mixture of methanol, acetonitrile, and water was identified to simultaneously extract multiple sphingolipids with broad polarity range. To reduce matrix effects, the targeted sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The extraction solvent was used as an isocratic mobile phase in chromatographic separation to eliminate solvent exchange steps and enable high-throughput multiple lipid assay. The assay is linear for ceramide from 0.6 to 9 μg/mL with bias <15 %. The intra-assay coefficient of variation is less than 10 % for concentrations from 1.2 to 9 μg/mL, and less than 25 % for concentrations below 1.2 μg/mL. For glucosylceramide and ceramide trihexoside the linear range is 0.05–3 μg/mL with biases <10 % and <20 %, respectively. The intra-assay coefficient of variation for these analytes is less than 10 % at concentrations from 0.4 to 3 μg/mL, and less than 25 % for concentrations below 0.4 μg/mL.
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Merrill AH Jr, Wang MD, Park M, Sullards MC (2007) (Glyco)sphingolipidology: an amazing challenge and opportunity for systems biology. Trends Biochem Sci 32:457–468
Patton JL, Lester RL (1991) The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane. J Bacteriol 173:3101–3108
Bielawski J, Pierce JS, Snider J, Rembiesa B, Szulc ZM, Bielawska A (2009) Comprehensive quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Methods Mol Biol 579:443–467
Hannun YA, Obeid LM (2002) The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J Biol Chem 277:25847–25850
Sastry PS (1985) Lipids of nervous tissue: composition and metabolism. Prog Lipid Res 24:69–176
Vos JP, Lopes-Cardozo M, Gadella BM (1994) Metabolic and functional aspects of sulfogalactolipids. Biochim Biophys Acta 1211:125–149
Giussani P, Tringali C, Riboni L, Viani P, Venerando B (2014) Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug resistance. Int J Mol Sci 15:4356–4392
Platt FM (2014) Sphingolipid lysosomal storage disorders. Nature 510:68–75
Ivleva VB, Sapp LM, O’Connor PB, Costello CE (2005) Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. J Am Soc Mass Spectrom 16:1552–1560
Gu M, Kerwin JL, Watts JD, Aebersold R (1997) Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry. Anal Biochem 244:347–356
Liebisch G, Drobnik W, Reil M, Trumbach B, Arnecke R, Olgemoller B, Roscher A, Schmitz G (1999) Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS). J Lipid Res 40:1539–1546
Mano N, Oda Y, Yamada K, Asakawa N, Katayama K (1997) Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry. Anal Biochem 244:291–300
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917
Lydic TA, Busik JV, Reid GE (2014) A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids. J Lipid Res 55:1797–1809
Bielawski J, Szulc ZM, Hannun YA, Bielawska A (2006) Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Methods 39:82–91
Little JL, Wempe MF, Buchanan CM (2006) Liquid chromatography-mass spectrometry/mass spectrometry method development for drug metabolism studies: examining lipid matrix ionization effects in plasma. J Chromatogr B Analyt Technol Biomed Life Sci 833:219–230
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Chuang, WL., Pacheco, J., Zhang, K. (2016). A Simple, High-Throughput Method for Analysis of Ceramide, Glucosylceramide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS. In: Garg, U. (eds) Clinical Applications of Mass Spectrometry in Biomolecular Analysis. Methods in Molecular Biology, vol 1378. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3182-8_28
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DOI: https://doi.org/10.1007/978-1-4939-3182-8_28
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3181-1
Online ISBN: 978-1-4939-3182-8
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