LC–MS/MS-Based System Used to Profile Ceramide Reactions to Diseases


Scientists from the University of Córdoba in Córdoba, Spain recently used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to comprehensively profile human ceramides to determine their reactions to diseases. Their findings were published in Analytica Chimica Acta (1).

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Ceramides are lipids characterized by a sphingoid base or backbone (SB) joined to a fatty acid (FA) via amide bonds. These lipids mostly hold structural functions in the cell membrane, though they are also useful as secondary messengers in cell differentiation, proliferation (promoted by very long-chain species) and apoptosis (induced by long-chain species). Recent studies have discussed ceramide modulation in pathological conditions, specifically proposing the use of them as potential biomarkers in different diseases. These were supported by findings of abnormal ceramide levels in blood for pathologies with different characteristics, such as ischemic stroke, multiple sclerosis, ovarian and colorectal cancer, Alzheimer’s disease, and coronary artery disease, among others.

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For this study, the scientists presented an overall strategy for this process, combining data-independent and dependent acquisitions (DIA and DDA, respectively) for the identification, confirmation, and quantitative determination of ceramides in human serum. With the application d of liquid chromatography–tandem mass spectrometry (LC–MS/MS) in DIA mode, 49 ceramides were identified, including d18:1, d18:0, d18:2, d16:1, d17:1 and t18:0 species. LC–MS/MS combines the separating power of liquid chromatography with the mass analysis capability of triple quadrupole mass spectrometry (2). For the quantitative determination of ceramides, a high-throughput and fully automated method made of solid-phase extraction on-line coupled to LC–MS/MS in DDA; this was done to improve analytical features and avoid errors associated with sample processing.

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Quantitation limits were recorded at the pg mL−1 level, with the intra-day and between-days variabilities being below 20% and 25%, respectively. The accuracy, which was expressed as bias, was always within ±25%. The proposed method was tested with a Coronary Diet Intervention with Olive Oil and Cardiovascular Prevention (CORDIOPREV) cohort in hopes of qualitatively and quantitatively profiling ceramides in human serum. This characterization showed d18:1 ceramides to be the most concentrated, with these ceramides making up 70.8% of the total concentration. This was followed by d18:2 and d18:0 with 13.0 % and 8.8 %, respectively. The less concentrated ceramides reportedly made up 7.1% of the total content.

This fragmentation pattern allowed a DIA method to be configured for the identification and confirmation of 49 ceramides, each of which were classified by the length, unsaturation grade and number of hydroxy groups of the SB, and the esterified FA. With these findings, it was concluded that combining DIA and DDA LC–MS/MS analysis enabled the scientists to qualitatively and quantitatively profile ceramides in human serum.


(1) Luque-Córdoba, D.; Calderón-Santiago, M.; Rangel-Zúñiga, O. A.; et al. Comprehensive Profiling of Ceramides in Human Serum by Liquid Chromatography Coupled to Tandem Mass Spectrometry Combining Data Independent/Dependent Acquisition Modes. Anal. Chim. Acta 2024, 1287, 342115. DOI: 10.1016/j.aca.2023.342115

(2) Liquid Chromatography Tandem Mass Spectrometry (LC–MS-MS). eUROFINS Scientific 2024. (accessed 2024-4-26)

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