Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography–electrospray ionization tandem mass spectrometry

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Abstract

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation and programmed cell death. Qualitative and quantitative analysis of these second messengers requires sensitive and specific analytical methods to detect endogenous levels of individual ceramide species and to differentiate between them. Nine synthetic ceramides were separated by liquid chromatography coupled to tandem mass spectrometry on a C18 bonded silica column. The lipids were eluted in gradient elution mode using a mixture of water, acetonitrile and 2-propanol as mobile phase. They were detected by reaction monitoring performed on positive ion electrospray generated ions. Collision-induced fragmentations conducted on ceramides produced a well characteristic product ion at m/z 264, making multiple reaction monitoring (MRM) well suited for various ceramides quantitative measurements. After optimization of the extraction step, the proposed methodology was able to identify and quantify different ceramide species issued from human cancer cells. The method could be validated for C16, C18 and C20 ceramides, quantified at the nanogram level. The validation exhibits good results with respect to linearity, accuracy and precision.

Introduction

In addition to their major structural role as a barrier for cell permeability and as a matrix for the association of membrane proteins, sphingolipids play a key role in the signal transduction and cell regulation.

Sphingomyelin hydrolysis to ceramides occurs through the action of sphingomyelin-specific forms of phospholipase C called sphingomyelinases. In addition to the breakdown of sphingomyelin to ceramide, a mechanism for the generation of ceramide exists through the action of ceramide synthase. Accurate and precise analytical methods, able to quantify selectively the various ceramide molecular species at low concentrations, are crucial for elucidating their function and metabolism.

Various experimental approaches have demonstrated that ceramides are key signaling molecules generated in response to a variety of stresses that mediate growth arrest, differentiation, senescence, apoptosis or an immune response [1], [2], [3], [4], [5], [6]. Nonetheless, their mechanisms of action remain largely unknown. In view of the increasing interest in sphingolipid metabolites, sensitive and specific methods are required to measure their endogenous levels.

Structurally, the ceramides exhibit a long amino alcoholic chain covalently bound via an amide linkage to a fatty acyl moiety (cf. Fig. 1). These molecules can vary in length, degree of unsaturation and hydroxylation, giving rise to a complex and diverse group of compounds. These lipids have proven to be very difficult to analyse due to their apolar nature, diversity and relatively low levels in biological samples.

Until now, the diacylglycerol (DAG) kinase assay is the most commonly used procedure for ceramide quantitation [7], [8]. The DAG kinase phosphorylates ceramide into a radioactive product which is detected by thin-layer chromatography (TLC). The principle limitations of the DAG kinase assay as well as the TLC measurement are their requirement of high sample volumes and their lack of discrimination capability between different ceramide species. Several other methods are also available for quantitation of sphingolipid metabolites by liquid chromatography (LC). Since the ceramides lack a chromophore group to allow a sensitive UV measurement, fluorescent or radioisotope detection after derivatization were perferred [9], [10], [11] All the above described procedures are time-consuming and tricky derivatization steps might often be source of error hampering the accurate determination of the ceramides levels. The use of an evaporative light-scattering detector for the ceramide visualization and identification provides direct analysis without any prior derivatization [12].

In the last few years, mass spectrometry methodologies have been developed for the detection of ceramides [13], [14], [15], [16], [17]. This technique is also a powerful tool for determining endogenous, physiologically active compounds because of its high selectivity and sensitivity. Electrospray ionization mass spectrometry (ESI-MS) has been successfully used for several years for the analysis of peptides and proteins and it has been more recently extended to the analysis of other species like carbohydrates and lipids [15], [17].

This work is focused on the separation and the identification of standard ceramides by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). The extraction procedure of lipids from human cells was optimized in order to obtain sufficient sensitivity. In human cancer cells, the endogenous ceramides were identified and quantitatively measured. Finally, the procedure was validated for C16, C18 and C20 ceramides and some analytical data are presented.

Section snippets

Chemicals

C2, C6, C8, C10, C12, C14, C16, C18 and C20 ceramides were obtained from Acros (Gell, Belgium).

Water, acetonitrile, 2-propanol, ethanol, formic acid were of HPLC grade from Merck (Darmstadt, Germany).

Nitrogen (alphagaz 1) and argon (alphagaz 2) were purchased from Air Liquide (Milmort, Belgium).

Instruments and methods

The high-performance liquid chromatograph (HPLC) is a HP 1100 series; it is equipped with a binary pump, a vacuum degasser, a thermostated column compartment and an autosampler, all from Agilent

Optimization of the MS detection

The MS analysis of ceramides was first investigated by direct introduction of the reference compounds dissolved in an ethanol–formic acid (99.8:0.2) mixture using the electrospray interface in the positive mode of ionization.

For each of the tested ceramide, two main peaks were observed corresponding respectively to the [M+H]+ and [M+H–H2O]+ ionic species (Fig. 1). Parameters such as the capillary and cone voltages, as well as the cone and the desolvation gas flows were optimized in order to

Conclusions

The method developed for the separation of ceramides present in human cancer cells using a LC–ESI-MS–MS procedure was found to be linear, accurate, precise and sensitive to the nanogram level.

This method enables accurate and precise analysis for ceramides issued from relatively small samples providing a useful tool for signal transduction research. In the future, this procedure will be used for studying the changes of endogenous ceramides concentrations that could occur during cellular stress

Acknowledgements

A research grant from the Belgium National Fund for Scientific Research (FNRS) to one of us (M.F.) is gratefully aknowledged. Many thanks are also due to FNRS for its financial support.

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