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Liquid chromatography-mass spectrometry for comprehensive profiling of ceramide molecules in human hair

2006; Elsevier BV; Volume: 47; Issue: 7 Linguagem: Inglês

10.1194/jlr.d600007-jlr200

1539-7262

Yoshinori Masukawa, Hisashi Tsujimura, Hirofumi Narita,

Lipid Membrane Structure and Behavior

Ceramides (CERs) play key roles in signal transduction and cell regulation, probably during the keratinization of human hair. Current methods using mass spectrometry (MS), however, are not sufficient to allow the comprehensive analysis of CER molecules, including isobaric and isomeric CERs. Therefore, a method for the comprehensive profiling of CERs was developed. The method developed is based on reversed-phase liquid chromatography (RPLC) coupled to atmospheric pressure chemical ionization (APCI)-MS. Comprehensive identification and profiling of CERs is achieved using two sets of multimass chromatograms obtained from two channel detections that monitor both molecular-related and sphingoid-related ions under two different in-source collision-induced dissociation conditions and using retention times obtained from RPLC. The application of this method revealed that human hair contains 73 species of CER molecules, which were all corroborated by structural analysis using tandem mass spectrometry. The results further revealed that the composition is characterized by predominant molecules consisting of even carbon atom-containing saturated/unsaturated nonhydroxy or α-hydroxy fatty acids and C18 dihydrosphingosine, a minor but distinct content of isobaric/isomeric and odd chain-containing CERs. This successfully developed RPLC-APCI-MS technique allows the comprehensive profiling of CER molecules in hair for the investigation of their physicochemical and physiological roles. Ceramides (CERs) play key roles in signal transduction and cell regulation, probably during the keratinization of human hair. Current methods using mass spectrometry (MS), however, are not sufficient to allow the comprehensive analysis of CER molecules, including isobaric and isomeric CERs. Therefore, a method for the comprehensive profiling of CERs was developed. The method developed is based on reversed-phase liquid chromatography (RPLC) coupled to atmospheric pressure chemical ionization (APCI)-MS. Comprehensive identification and profiling of CERs is achieved using two sets of multimass chromatograms obtained from two channel detections that monitor both molecular-related and sphingoid-related ions under two different in-source collision-induced dissociation conditions and using retention times obtained from RPLC. The application of this method revealed that human hair contains 73 species of CER molecules, which were all corroborated by structural analysis using tandem mass spectrometry. The results further revealed that the composition is characterized by predominant molecules consisting of even carbon atom-containing saturated/unsaturated nonhydroxy or α-hydroxy fatty acids and C18 dihydrosphingosine, a minor but distinct content of isobaric/isomeric and odd chain-containing CERs. This successfully developed RPLC-APCI-MS technique allows the comprehensive profiling of CER molecules in hair for the investigation of their physicochemical and physiological roles. Ceramides (CERs), which consist of a long amino alcoholic chain [sphingoid moiety (SPM)] covalently bound via an amide linkage to a fatty acid moiety (FAM), have important physicochemical roles in the barrier function and water-holding property of skin, together with other lipids in intercellular spaces of the stratum corneum (1Elias P.M. Epidermal lipids, barrier, function, and desquamation.J. Invest. Dermatol. 1983; 80: 44-49Abstract Full Text PDF PubMed Google Scholar, 2Imokawa G. Akasaki S. Hattori M. Yoshizuka N. Selective recovery of deranged water-holding properties by stratum corneum.J. Invest. Dermatol. 1986; 87: 758-761Abstract Full Text PDF PubMed Google Scholar). They also play key roles in signal transduction and cell regulation relevant to cell growth arrest, differentiation, senescence, apoptosis, and immune responses (3Fishbein J.D. Dobrowsky R.T. Bielawska A. Garrett S. Hannun Y.A. Ceramide-mediated growth inhibition and CAPP are conserved in Saccharomyces cerevisiae.J. Biol. Chem. 1993; 268: 9244-9261Abstract Full Text PDF Google Scholar, 4Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hunnun Y.A. Role for ceramide in cell cycle arrest.J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 5Riboni L. Prinetti A. Bassi R. Caminiti A. Tettamanti G. A mediator role of ceramide in the regulation of neuroblastoma Neuro2a cell differentiation.J. Biol. 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Human cells/tissues predominantly include CERs consisting of an even carbon atom-containing nonhydroxy FAM and a C18 sphingosine moiety together with less abundant CERs consisting of an even carbon atom-containing α-hydroxy FAM and a C18 sphingosine moiety (10Yamada Y. Kajiwara K. Yano M. Kishida E. Masuzawa Y. Kojo S. Increase of ceramides and its inhibition by catalase during chemically induced apoptosis of HL-60 cells determined by electrospray ionization tandem mass spectrometry.Biochim. Biophys. Acta. 2001; 1532: 115-120Crossref PubMed Scopus (20) Google Scholar, 11Fillet M. Van Heugen J-C. Servais A-C. De Graeve J. Crommen J. Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography-electrospray ionization tandem mass spectrometry.J. Chromatogr. A. 2002; 949: 225-233Crossref PubMed Scopus (43) Google Scholar, 12Sullards M.C. Wang E. Peng Q. Merril Jr., A.H. Metabolomic profiling of sphingolipids in human glioma cell lines by liquid chromatography tandem mass spectrometry.Cell. Mol. Biol. 2003; 49: 789-797PubMed Google Scholar, 13Drobnik W. Liebisch G. Audebert F-X. Froählich D. Gluäck T. Vogel P. Rothe G. Schmitz G. Plasma ceramide and lysophosphatidylcholine inversely correlate with mortality in sepsis patients.J. Lipid Res. 2003; 44: 754-761Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 14Merrill A.H. Sweeley C.C. Sphingolipids: metabolism and cell signaling.in: Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, New York1996: 309-339Crossref Scopus (12) Google Scholar, 15Alderson N.L. Walla M.D. Hama H. A novel method for the measurement of in vivo fatty acid 2-hydroxylase activity by gas chromatography-mass spectrometry.J. Lipid Res. 2005; 46: 1569-1575Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Among these CERs, it is thought that the molecules with C16 or C24 nonhydroxy FAMs may be relevant to apoptosis (16Thomas R.L. Matsko C.M. Lotze M.T. Amoscato A.A. Mass spectrometric identification of increased C16 ceramide levels during apoptosis.J. Biol. Chem. 1999; 274: 30580-30588Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 17Kroesen B-J. Pettus B. Luberto C. Busman M. Sietsma H. De Leji L. Hannun Y.A. Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria.J. Biol. Chem. 2001; 276: 13606-13614Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 18Kroesen B-J. Jacobs S. Pettus B.J. Sietsma H. Kok J.W. Hannun Y.A. De Leji L. F. N.H. BcR-induced apoptosis involves differential regulation of C16 and C24 ceramide formation and sphingolipid-dependent activation of the proteasome.J. Biol. Chem. 2003; 278: 14723-14731Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The possibility that specific CER molecules may be involved with human diseases such as sphingolipidosis and peroxisomal disorders has also been proposed (19Fujiwaki T. Yamaguchi S. Sukegawa K. Taketomi T. Application of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in tissues from sphingolipidosis patients.J. Chromatogr. B. 1999; 731: 45-52Crossref PubMed Scopus (33) Google Scholar, 20Pettus B.J. Baes M. Busman M. Hannun Y.A. Van Veldhoven P.P. Mass spectrometric analysis of ceramide perturbations in brain and fibroblast of mice and human patients with peroxisomal disorders.Rapid Commun. Mass Spectrom. 2004; 18: 1569-1574Crossref PubMed Scopus (39) Google Scholar). Although only predominant CERs are currently targeted, less abundant but bioactive CERs should also be comprehensively studied. There are extremely complex CERs consisting of nonhydroxy, α-hydroxy, or ω-hydroxy FAMs and SPMs, such as sphingosine, dihydrosphingosine, phytosphingosine, or 6-hydroxy-sphingosine, in human stratum corneum (21Robson K.J. Stewart M.E. Michelsen S. Lazo N.D. Downing D.T. 6-Hydroxy-4-sphingenine in human epidermal ceramides.J. Lipid Res. 1994; 35: 2060-2068Abstract Full Text PDF PubMed Google Scholar). Some dermatologists are interested in the dynamics of CER-related lipids in differentiated keratinocytes where apoptosis occurs in skin (22Hamanaka S. Nakazawa S. Yamanaka M. Uchida Y. Otsuka F. Glucosylceramide accumulates preferentially in lamella bodies in differentiated keratinocytes.Br. J. Dermatol. 2005; 152: 426-434Crossref PubMed Scopus (40) Google Scholar). On the other hand, human hair also includes CERs that are predominantly composed of a nonhydroxy or α-hydroxy FAM and a dihydrosphingosine moiety (23Hussler G. Kaba G. Francois A.M. Saint-Leger D. Isolation and identification of human hair ceramides.Int. J. Cosmet. Sci. 1995; 17: 197-206Crossref PubMed Scopus (19) Google Scholar, 24Masukawa Y. Narita H. Imokawa G. Characterization of the lipid composition at the proximal root regions of human hair.J. Cosmet. Sci. 2005; 56: 1-16PubMed Google Scholar). We hypothesize that the CERs may be related to apoptosis during keratinization that proceeds from living hair matrix cells to dead cuticular or cortical cells, ascribed to their bioactivities, and may contribute to barrier function and water holding in hair. To drive such a study, we need an analytical method for the comprehensive profiling of all CER molecules in hair, including not only the predominant CERs but also isobaric CERs and less abundant α-hydroxy FAM-containing CERs. Many methods, such as thin-layer chromatography (25Imokawa G. Abe A. Jin K. Higaki Y. Kawashima M. Hidano A. Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin.J. Invest. Dermatol. 1991; 96: 523-526Abstract Full Text PDF PubMed Scopus (822) Google Scholar), diacylglycerol kinase assay (26Van Veldhoven P.P. Bishop W.R. Yurivich D.A. Bell R.M. Ceramide quantitation: evaluation of a mixed micellar assay using E. coli diacylglycerol kinase.Biochem. Mol. Biol. Int. 1995; 36: 21-30PubMed Google Scholar), liquid chromatography (LC) connected to evaporative light-scattering detection (27McNabb T.J. Cremesti A.E. Brown P.R. Fischl A.S. The separation and direct detection of ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and evaporative light-scattering detection.Anal. Biochem. 1999; 276: 242-250Crossref PubMed Scopus (30) Google Scholar), and LC connected to ultraviolet light detection using a derivatizing technique (28Hoi U. Pei P.T. Minard R.D. Separation of molecular species of ceramides as benzoyl and p-nitrobenzoyl derivatives by high performance liquid chromatography.Lipids. 1981; 16: 855-862Crossref Scopus (17) Google Scholar), have been used to analyze CERs. However, these conventional methods are not sufficient to comprehensively detect CER molecules in biological materials, despite being useful for the determination of total, type, and/or molecular levels of interesting CERs. Although gas chromatography-mass spectrometry (MS) can individually detect trimethylsilylated CERs (23Hussler G. Kaba G. Francois A.M. Saint-Leger D. Isolation and identification of human hair ceramides.Int. J. Cosmet. Sci. 1995; 17: 197-206Crossref PubMed Scopus (19) Google Scholar, 24Masukawa Y. Narita H. Imokawa G. Characterization of the lipid composition at the proximal root regions of human hair.J. Cosmet. Sci. 2005; 56: 1-16PubMed Google Scholar), it cannot comprehensively detect minor CERs as well as major CERs because of its lower sensitivity. Since Mano et al. (29Mano N. Oda Y. Yamada K. Asakawa N. Katayama K. Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry.Anal. Biochem. 1997; 244: 291-300Crossref PubMed Scopus (123) Google Scholar), Gu et al. (30Gu M. Kerwin J.L. Watts J.D. Aebersold R. Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry.Anal. Biochem. 1997; 244: 347-356Crossref PubMed Scopus (127) Google Scholar), and Couch et al. (31Couch L.H. Churchwell M.I. Doerge D.R. Tolleson W.H. Howard P.C. Identification of ceramides in human cells using liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry.Rapid Commun. Mass Spectrom. 1997; 11: 504-512Crossref PubMed Google Scholar) demonstrated MS using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) to be extremely valuable tools in the analysis of mixed CER molecules, many methods for the identification and determination of CER molecules have been proposed and applied to the analysis of CERs in human cells/tissues (10Yamada Y. Kajiwara K. Yano M. Kishida E. Masuzawa Y. Kojo S. Increase of ceramides and its inhibition by catalase during chemically induced apoptosis of HL-60 cells determined by electrospray ionization tandem mass spectrometry.Biochim. Biophys. Acta. 2001; 1532: 115-120Crossref PubMed Scopus (20) Google Scholar, 32Raith K. Neubert R.H.H. Structural studies on ceramides by electrospray tandem mass spectrometry.Rapid Commun. Mass Spectrom. 1998; 12: 935-938Crossref Scopus (42) Google Scholar, 33Liebisch G. Drobnik W. Reil M. Truämbach B. Arneche R. Olgemoäller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 34Raith K. Zellmer S. Lasch J. Neubert R.H.H. Profiling of human stratum corneum ceramides by liquid chromatography-electrospray tandem mass spectrometry.Anal. Chim. Acta. 2000; 418: 167-173Crossref Scopus (28) Google Scholar, 35Vietzke J-P. Brandt O. Abeck D. Rapp C. Strassner M. Schreiner V. Hinze U. Comparative investigation of human stratum corneum ceramides.Lipids. 2001; 36: 299-304Crossref PubMed Scopus (44) Google Scholar, 36Hsu F-F. Turk J. Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 558-570Crossref PubMed Scopus (88) Google Scholar, 38Han X. Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2002; 302: 199-212Crossref PubMed Scopus (148) Google Scholar, 37Hsu F-F. Turk J. Stewart M.E. Downing D.T. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 680-695Crossref PubMed Scopus (82) Google Scholar, 39Lee M.H. Lee G.H. Yoo J.S. Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation.Rapid Commun. Mass Spectrom. 2003; 17: 64-75Crossref PubMed Scopus (43) Google Scholar, 40Yamane M. Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography-thermospray mass spectrometry.J. Chromatogr. B. 2003; 783: 181-190Crossref PubMed Scopus (15) Google Scholar, 41Farwanah H. Nuhn P. Neubert R. Raith K. Normal-phase liquid chromatographic separation of stratum corneum ceramides with detection by evaporative light scattering and atmospheric pressure chemical ionization mass spectrometry.Anal. Chim. Acta. 2003; 492: 233-239Crossref Scopus (21) Google Scholar, 42Pettus B.J. Bielawska A. Kroesen B-J. Moeller P.D.R. Szulc Z.M. Hannun Y.A. Busman M. Observation of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 17: 1203-1211Crossref PubMed Scopus (38) Google Scholar, 43Pettus B.J. Kroesen B-J. Szulc Z.M. Bielawska A. Bielawski J. Hannun Y.A. Busman M. Quantitative measurement of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 18: 577-583Crossref Scopus (45) Google Scholar, 44Sullards M.C. Wang E. Peng Q. Merril Jr., A.H. Metabolomic profiling of sphingolipids in human glioma cell lines by liquid chromatography tandem mass spectrometry.Cell. Mol. Biol. 2003; 49: 789-797PubMed Google Scholar, 45Farwanah H. Raith K. Neubert R.H.H. Wohlrab J. Ceramide profiles of the uninvolved skin in atopic dermatitis and psoriasis are comparable to those of healthy skin.Arch. Dermatol. Res. 2005; 296: 514-521Crossref PubMed Scopus (93) Google Scholar): reversed-phase liquid chromatography (RPLC) (34Raith K. Zellmer S. Lasch J. Neubert R.H.H. Profiling of human stratum corneum ceramides by liquid chromatography-electrospray tandem mass spectrometry.Anal. Chim. Acta. 2000; 418: 167-173Crossref Scopus (28) Google Scholar, 35Vietzke J-P. Brandt O. Abeck D. Rapp C. Strassner M. Schreiner V. Hinze U. Comparative investigation of human stratum corneum ceramides.Lipids. 2001; 36: 299-304Crossref PubMed Scopus (44) Google Scholar, 39Lee M.H. Lee G.H. Yoo J.S. Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation.Rapid Commun. Mass Spectrom. 2003; 17: 64-75Crossref PubMed Scopus (43) Google Scholar, 40Yamane M. Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography-thermospray mass spectrometry.J. Chromatogr. B. 2003; 783: 181-190Crossref PubMed Scopus (15) Google Scholar), normal-phase LC (41Farwanah H. Nuhn P. Neubert R. Raith K. Normal-phase liquid chromatographic separation of stratum corneum ceramides with detection by evaporative light scattering and atmospheric pressure chemical ionization mass spectrometry.Anal. Chim. Acta. 2003; 492: 233-239Crossref Scopus (21) Google Scholar, 42Pettus B.J. Bielawska A. Kroesen B-J. Moeller P.D.R. Szulc Z.M. Hannun Y.A. Busman M. Observation of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 17: 1203-1211Crossref PubMed Scopus (38) Google Scholar, 43Pettus B.J. Kroesen B-J. Szulc Z.M. Bielawska A. Bielawski J. Hannun Y.A. Busman M. Quantitative measurement of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 18: 577-583Crossref Scopus (45) Google Scholar, 45Farwanah H. Raith K. Neubert R.H.H. Wohlrab J. Ceramide profiles of the uninvolved skin in atopic dermatitis and psoriasis are comparable to those of healthy skin.Arch. Dermatol. Res. 2005; 296: 514-521Crossref PubMed Scopus (93) Google Scholar) connected to single quadrupole MS (39Lee M.H. Lee G.H. Yoo J.S. Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation.Rapid Commun. Mass Spectrom. 2003; 17: 64-75Crossref PubMed Scopus (43) Google Scholar, 40Yamane M. Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography-thermospray mass spectrometry.J. Chromatogr. B. 2003; 783: 181-190Crossref PubMed Scopus (15) Google Scholar, 44Sullards M.C. Wang E. Peng Q. Merril Jr., A.H. Metabolomic profiling of sphingolipids in human glioma cell lines by liquid chromatography tandem mass spectrometry.Cell. Mol. Biol. 2003; 49: 789-797PubMed Google Scholar) or ion trap MS (34Raith K. Zellmer S. Lasch J. Neubert R.H.H. Profiling of human stratum corneum ceramides by liquid chromatography-electrospray tandem mass spectrometry.Anal. Chim. Acta. 2000; 418: 167-173Crossref Scopus (28) Google Scholar, 35Vietzke J-P. Brandt O. Abeck D. Rapp C. Strassner M. Schreiner V. Hinze U. Comparative investigation of human stratum corneum ceramides.Lipids. 2001; 36: 299-304Crossref PubMed Scopus (44) Google Scholar, 41Farwanah H. Nuhn P. Neubert R. Raith K. Normal-phase liquid chromatographic separation of stratum corneum ceramides with detection by evaporative light scattering and atmospheric pressure chemical ionization mass spectrometry.Anal. Chim. Acta. 2003; 492: 233-239Crossref Scopus (21) Google Scholar, 42Pettus B.J. Bielawska A. Kroesen B-J. Moeller P.D.R. Szulc Z.M. Hannun Y.A. Busman M. Observation of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 17: 1203-1211Crossref PubMed Scopus (38) Google Scholar, 43Pettus B.J. Kroesen B-J. Szulc Z.M. Bielawska A. Bielawski J. Hannun Y.A. Busman M. Quantitative measurement of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 18: 577-583Crossref Scopus (45) Google Scholar, 45Farwanah H. Raith K. Neubert R.H.H. Wohlrab J. Ceramide profiles of the uninvolved skin in atopic dermatitis and psoriasis are comparable to those of healthy skin.Arch. Dermatol. Res. 2005; 296: 514-521Crossref PubMed Scopus (93) Google Scholar), and direct triple quadrupole MS (10Yamada Y. Kajiwara K. Yano M. Kishida E. Masuzawa Y. Kojo S. Increase of ceramides and its inhibition by catalase during chemically induced apoptosis of HL-60 cells determined by electrospray ionization tandem mass spectrometry.Biochim. Biophys. Acta. 2001; 1532: 115-120Crossref PubMed Scopus (20) Google Scholar, 32Raith K. Neubert R.H.H. Structural studies on ceramides by electrospray tandem mass spectrometry.Rapid Commun. Mass Spectrom. 1998; 12: 935-938Crossref Scopus (42) Google Scholar, 33Liebisch G. Drobnik W. Reil M. Truämbach B. Arneche R. Olgemoäller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 36Hsu F-F. Turk J. Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 558-570Crossref PubMed Scopus (88) Google Scholar, 37Hsu F-F. Turk J. Stewart M.E. Downing D.T. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 680-695Crossref PubMed Scopus (82) Google Scholar, 38Han X. Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2002; 302: 199-212Crossref PubMed Scopus (148) Google Scholar). However, these methods do not provide comprehensive profiling of all CER molecules, including isobaric and isomeric CERs. Furthermore, it is unknown whether those methods are optimal to detect lower levels of α-hydroxy FAM-containing CERs that are present in human cells/tissues, because those CERs were not detected in any of their applications (10Yamada Y. Kajiwara K. Yano M. Kishida E. Masuzawa Y. Kojo S. Increase of ceramides and its inhibition by catalase during chemically induced apoptosis of HL-60 cells determined by electrospray ionization tandem mass spectrometry.Biochim. Biophys. Acta. 2001; 1532: 115-120Crossref PubMed Scopus (20) Google Scholar, 32Raith K. Neubert R.H.H. Structural studies on ceramides by electrospray tandem mass spectrometry.Rapid Commun. Mass Spectrom. 1998; 12: 935-938Crossref Scopus (42) Google Scholar, 33Liebisch G. Drobnik W. Reil M. Truämbach B. Arneche R. Olgemoäller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 34Raith K. Zellmer S. Lasch J. Neubert R.H.H. Profiling of human stratum corneum ceramides by liquid chromatography-electrospray tandem mass spectrometry.Anal. Chim. Acta. 2000; 418: 167-173Crossref Scopus (28) Google Scholar, 35Vietzke J-P. Brandt O. Abeck D. Rapp C. Strassner M. Schreiner V. Hinze U. Comparative investigation of human stratum corneum ceramides.Lipids. 2001; 36: 299-304Crossref PubMed Scopus (44) Google Scholar, 36Hsu F-F. Turk J. Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 558-570Crossref PubMed Scopus (88) Google Scholar, 37Hsu F-F. Turk J. Stewart M.E. Downing D.T. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 680-695Crossref PubMed Scopus (82) Google Scholar, 38Han X. Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2002; 302: 199-212Crossref PubMed Scopus (148) Google Scholar, 39Lee M.H. Lee G.H. Yoo J.S. Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation.Rapid Commun. Mass Spectrom. 2003; 17: 64-75Crossref PubMed Scopus (43) Google Scholar, 40Yamane M. Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography-thermospray mass spectrometry.J. Chromatogr. B. 2003; 783: 181-190Crossref PubMed Scopus (15) Google Scholar, 41Farwanah H. Nuhn P. Neubert R. Raith K. Normal-phase liquid chromatographic separation of stratum corneum ceramides with detection by evaporative light scattering and atmospheric pressure chemical ionization mass spectrometry.Anal. Chim. Acta. 2003; 492: 233-239Crossref Scopus (21) Google Scholar, 42Pettus B.J. Bielawska A. Kroesen B-J. Moeller P.D.R. Szulc Z.M. Hannun Y.A. Busman M. Observation of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 17: 1203-1211Crossref PubMed Scopus (38) Google Scholar, 43Pettus B.J. Kroesen B-J. Szulc Z.M. Bielawska A. Bielawski J. Hannun Y.A. Busman M. Quantitative measurement of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 18: 577-583Crossref Scopus (45) Google Scholar, 44Sullards M.C. Wang E. Peng Q. Merril Jr., A.H. Metabolomic profiling of sphingolipids in human glioma cell lines by liquid chromatography tandem mass spectrometry.Cell. Mol. Biol. 2003; 49: 789-797PubMed Google Scholar, 45Farwanah H. Raith K. Neubert R.H.H. Wohlrab J. Ceramide profiles of the uninvolved skin in atopic dermatitis and psoriasis are comparable to those of healthy skin.Arch. Dermatol. Res. 2005; 296: 514-521Crossref PubMed Scopus (93) Google Scholar), despite their successful detection in authentic samples. The aim of this study was to develop a method for the comprehensive profiling of CER molecules using LC-MS. Consequently, this method was successfully developed by optimizing RPLC connected to APCI-MS in the positive ion mode, which uses two channel detections that monitor both molecular-related and SPM-related ions under two different in-source collision-induced dissociation (CID) conditions. Here, we show that the composition of CERs in hair is characterized not only by predominant CER molecules consisting of saturated/unsaturated FAMs with even numbers of carbon atoms and a C18 dihydrosphingosine moiety but also by isobaric or isomeric CERs, α-hydroxy fatty acid-containing CERs, and odd chain-containing CERs. CERs are termed according to Motta et al. (46Motta S. Monti M. Sesana S. Caputo R. Carelli S. Ghid