Highly Sensitive Determination of Diverse Ceramides in Human Hair Using Reversed‐Phase High‐Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry

Masukawa, Yoshinori; Tsujimura, Hiroji

doi:10.1007/s11745-006-3012-6

Cited by 13 publications

(13 citation statements)

References 44 publications

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“…5A . Subsequently, MS/MS experiments were conducted on CER [N (24) injected using an identical setup as was used for synthetic CERs. The CER total ion chromatogram of the human SC sample is shown in Fig.…”

Section: Because No Synthetic Variant Of Cer [Eods] Is Commercially Amentioning

confidence: 99%

“…However, these approaches are very time consuming and a high quantity of material is required, which is not always available. The application of LC/MS results in a much more detailed profi le of each individual subclass using only small quantities of material (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Besides their important role in human skin, CERs are also key molecules with respect to cell signaling, growth, differentiation, and apoptosis ( 29-38 ).…”

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confidence: 99%

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LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Smeden

Hoppel²,

Heijden³

et al. 2011

Journal of Lipid Research

202

149

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organization of these lipids are of major importance for a competent skin barrier function ( 1-4 ). The major lipid classes in human SC are cholesterol, free fatty acids, and ceramides (CERs). In particular, the CERs have drawn much attention and several reported studies demonstrate that changes in CER composition may play a role in an impaired skin barrier ( 5-11 ). The various CERs consist of a sphingoid base linked via an amide bond to a fatty acid. Both variations in the fatty acid carbon chain and the sphingoid base architecture result in a large number of CER subclasses with a wide variation in chain length distribution. The molecular architecture of these subclasses is depicted in Fig. 1 . Over the years, important information about CER composition in human SC has been obtained using high performance thin layer chromatography (HPTLC) in conjunction with NMR. This resulted in a gradual increase in the number of identifi ed subclasses. By use of these methods, nine different human CER subclasses have been identifi ed in the SC of healthy human skin ( 12-16 ). As HPTLC is easily accessible, it is still frequently used in various fi elds with respect to skin lipid research to determine the CER subclasses in human SC ( 11,17,18 ). However, these approaches are very time consuming and a high quantity of material is required, which is not always available. The application of LC/MS results in a much more detailed profi le of each individual subclass using only small quantities of material (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Besides their important role in human skin, CERs are also key molecules with respect to cell signaling, growth, differentiation, and apoptosis ( 29-38 ). Also in these research areas, the introAbstract Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identifi ed. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identifi ed subclasses, a 3D multimass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked -hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identifi cation of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from fullthickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profi le. The outermost layer of mammalian skin, the stratum corneum (SC), consists of corneocytes embedd...

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Section: Because No Synthetic Variant Of Cer [Eods] Is Commercially Amentioning

confidence: 99%

mentioning

confidence: 99%

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Smeden

Hoppel²,

Heijden³

et al. 2011

Journal of Lipid Research

202

149

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“…Provided that the LOD and LOQ are determined according to the S/N of 3 and 10, respectively, our method would have almost the same LOQ as that method. The other method ( 46 ), which uses RPLC-ESI-MS, has an LOQ of 0.48-1.37 fmol for 13 authentic CER[NDS], CER[NS], CER[ADS] and CER[AS] that exist in human hair, which indicates higher sensitivity of that method than our method. However, it should be noted that in both those methods, the targeted species are restricted to 2 or 4 classes.…”

Section: Discussionmentioning

confidence: 91%

Comprehensive quantification of ceramide species in human stratum corneum

Masukawa

Narita

Sato

et al. 2009

Journal of Lipid Research

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150

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This article is available online at http://www.jlr.org novo pathways, such as alterations of some species of the targeted lipidomes into others. Similar to common quantitative analyses using direct MS or LC-MS that need authentic standards together with internal ones, quantitative lipidomics may also require a standard set of all lipid species targeted, because each lipid species has a different molar response in MS detection except for special cases. However, it is practically impossible to obtain all authentic species, including not only different even straight carbon chains but also odd and/or branched ones. To overcome such limitations, lipidomics researchers have contrived a novel procedure to quantify lipidomes comprehensively. Han and Gross ( 3 ) proposed a quantitative method for analyzing triglyceride molecular species using direct MS, which involves a procedure to calculate response factors for species that are not available as authentic materials, based on differences of the responses in the numbers of total carbons and double bonds among the species. For quantitative determination of phospholipid molecular species, Koivusalo et al. ( 4 ) utilized the following parameAbstract One of the key challenges in lipidomics is to quantify lipidomes of interest, as it is practically impossible to collect all authentic materials covering the targeted lipidomes. For diverse ceramides (CER) in human stratum corneum (SC) that play important physicochemical roles in the skin, we developed a novel method for quantifi cation of the overall CER species by improving our previously reported profi ling technique using normal-phase liquid chromatography-electrospray ionization-mass spectrometry (NPLC-ESI-MS). The use of simultaneous selected ion monitoring measurement of as many as 182 kinds of molecular-related ions enables the highly sensitive detection of the overall CER species, as they can be analyzed in only one SC-stripped tape as small as 5 mm × 10 mm. To comprehensively quantify CERs, including those not available as authentic species, we designed a procedure to estimate their levels using relative responses of representative authentic species covering the species targeted, considering the systematic error based on intra-/inter-day analyses. The CER levels obtained by this method were comparable to those determined by conventional thin-layer chromatography (TLC), which guarantees the validity of this method. , ceramide class consisting of non-hydroxy fatty acids and 4-sphingenines; ESI, electrospray ionization; LOD, limit of detection; LOQ, limit of quantifi cation; NPLC, normal-phase liquid chromatography; RPLC, reversed-phase liquid chromatography; RSD, relative standard deviation; SC, stratum corneum; SIM, selected ion monitoring; S/N, signal to noise; TLC, thinlayer chromatography

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“…19−21 In accordance with the literature, CERs can be detected in both ionization modes. 1,22,23 For measurements in negative ESI, methanol extracts can be subjected onto the LC-MS methodology without further pretreatment apart from concentration via evaporation. Solid-phase extraction (SPE), however, is a definite requirement when analyzing skin CER samples in positive ESI mode.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Profiling and Characterizing Skin Ceramides Using Reversed-Phase Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

t’Kindt¹,

Jorge²,

et al. 2011

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An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).

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Highly Sensitive Determination of Diverse Ceramides in Human Hair Using Reversed‐Phase High‐Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry

Cited by 13 publications

References 44 publications

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Comprehensive quantification of ceramide species in human stratum corneum

Profiling and Characterizing Skin Ceramides Using Reversed-Phase Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

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