Normal phase liquid chromatography coupled to quadrupole time of flight atmospheric pressure chemical ionization mass spectrometry for separation, detection and mass spectrometric profiling of neutral sphingolipids and cholesterol

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 877; no. 27; pp. 2976 - 2982
• Main Authors: Farwanah, Hany, Wirtz, Jennifer, Kolter, Thomas, Raith, Klaus, Neubert, Reinhard H.H., Sandhoff, Konrad
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-10-2009; Elsevier
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; APCI-MS; Biological and medical sciences; Cells, Cultured; Cholesterol; Cholesterol - analysis; Cholesterol - isolation & purification; Chromatography, High Pressure Liquid - methods; Chromatography, Thin Layer - methods; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; General pharmacology; HPLC/MS; HPTLC; Humans; Lipidomics; Mass Spectrometry - methods; Medical sciences; Neutral Glycosphingolipids - analysis; Neutral Glycosphingolipids - isolation & purification; Neutral sphingolipids; Pharmacology. Drug treatments; Q-TOF; Reproducibility of Results; Sensitivity and Specificity; Sphingolipidoses; Sphingolipidoses; HPLC/MS; APCI-MS; Q-TOF; Lipidomics; Neutral sphingolipids; Cholesterol; HPTLC; Coupled method; HPLC chromatography; Sphingolipid; Lipids; Quadrupole spectrometer; Thin layer chromatography; Normal phase chromatography; Time of flight method; Detection; Mass spectrometry
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.07.008

Many lipidomic approaches focus on investigating aspects of sphingolipid metabolism. Special emphasis is put on neutral sphingolipids and cholesterol and their interaction. Such an interest is attributed to the fact that those lipids are altered in a series of serious disorders including various sphingolipidoses. High performance thin-layer chromatography (HPTLC) has become a widely used technique for lipid analysis. However, mass spectrometric profiling is irreplaceable for gaining an overview about the various molecular species within a lipid class. In this work we have developed a sensitive method based on a gradient normal phase high performance liquid chromatography (HPLC) coupled to quadrupole time of flight (QTOF) atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in positive mode, which for the first time enables separation, on-line detection, and mass spectrometric profiling of multiple neutral sphingolipids including ceramide, glucosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin as well as cholesterol within less than 15
min. An important advantage of the presented HPLC/APCI-MS approach is that the separation pattern emulates the one obtained by an optimized HPTLC method with a multiple stage development. Thus, the lipid classes previously separated and quantified by HPTLC can be easily screened regarding their mass spectrometric profiles by HPLC/APCI-MS. In addition, the selected ionization conditions enable in-source fragmentation providing useful structural information. The methods (HPLC/APCI-MS and the optimized HPTLC) were applied for the analysis of the mentioned lipids in human fibroblasts. This approach is aimed basically at investigators who perform studies based on genetic modifications or treatment with pharmacological agents leading to changes in the biochemical pathways of neutral sphingolipids and cholesterol. In addition, it can be of interest for research on disorders related to impairments of sphingolipid metabolism.