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

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Lipids

Abstract

Since ceramides (CERs) play roles in signal transduction and cell regulation, CERs of human hair might be responsible for apoptosis during keratinization, in addition to their structural barrier and water-holding functions. Although, we previously developed a method for comprehensive profiling of the CERs in hair, that method was too insensitive to quantitatively characterize the CERs in a small amount of hair samples. The aim of this study was to develop a novel method for the highly sensitive determination of the diverse CERs. The method developed is negative ion electrospray ionization mass spectrometry (ESI-MS) coupled to reversed-phase high-performance liquid chromatography (RP-HPLC) using methanol containing 10 mM ammonium acetate as a mobile phase. By this method, 48 peaks derived from 73 kinds of CERs were simultaneously determined in selected ion monitoring measurement using one calibration line of the standard N-palmitoyl dihydrosphigosine, based on extremely small differences in the molar responses among different species of CERs, followed by the calculation of the actual levels using corrections for 13C and 2H effects. This method had extremely high sensitivity as indicated in the limit of quantification being in the femtomolar range. Other quantitative validation data, such as reproducibility, linearity and recoveries, were all sufficient. The quantitative levels of CERs determined by RP-HPLC–ESI-MS were comparable with those determined by thin-layer chromatography. This method was successfully applied to the characterization of levels of CERs in only 1-mm pieces derived from a single hair fiber and revealed the presence of interindividual and intraindividual variations of the CER composition. This RP-HPLC–ESI-MS method can be a powerful tool for future research on physicochemical and physiological roles of CERs in hair.

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Abbreviations

% CV:

Percentage coefficient of variation

ADS:

Ceramides consisting of α-hydroxy fatty acid and dihydrosphingosine moieties

APCI:

Atmospheric pressure chemical ionization

AS:

Ceramides consisting of α-hydroxy fatty acid and sphingosine moieties

CER:

Ceramide

DAG:

Diacylglycerol

ESI:

Electrospray ionization

LC:

Liquid chromatography

LOD:

Limit of detection

LOQ:

Limit of quantification

MS:

Mass spectrometry

NDS:

Ceramides consisting of non-hydroxy fatty acid and dihydrosphingosine moieties

NS:

Ceramides consisting of non-hydroxy fatty acid and sphingosine moieties

RP-HPLC:

Reversed-phase high-performance liquid chromatography

SIM:

Selected ion monitoring

TLC:

Thin-layer chromatography

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Acknowledgements

We would like to express our cordial gratitude to Katsumi Kita of the Kao Corporation for his discussions and encouragement of this study. Our sincere thanks also go to Manabu Watanabe, Yoshiya Sugai and Yoshinori Nishizawa of the Kao Corporation for technical support in the synthesis of standard CERs.

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Authors and Affiliations

  1. Tochigi Research Laboratories, Kao Corporation, Ichikai, Haga, Tochigi, 321-3497, Japan

    Yoshinori Masukawa & Hisashi Tsujimura

  2. Analytical Research Center, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi, 321-3497, Japan

    Yoshinori Masukawa

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  1. Yoshinori Masukawa
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  2. Hisashi Tsujimura
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Correspondence to Yoshinori Masukawa.

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Masukawa, Y., Tsujimura, H. Highly Sensitive Determination of Diverse Ceramides in Human Hair Using Reversed-Phase High-Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry. Lipids 42, 275–290 (2007). https://doi.org/10.1007/s11745-006-3012-6

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  • DOI: https://doi.org/10.1007/s11745-006-3012-6

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