LipidQuan: HILIC-Based LC-MS/MS High-Throughput Targeted Ceramide and Hexosylceramide Screen

Significance of the Topic

The targeted analysis of sphingolipids is vital for elucidating cellular signaling, membrane integrity, and disease mechanisms. Ceramides and hexosylceramides serve as bioactive lipids involved in apoptosis, tumor proliferation, chemotherapeutic resistance, and neurodegenerative disorders such as Parkinson’s disease. A rapid, reliable workflow for quantifying these lipids in plasma and serum supports biomarker discovery, drug development, and clinical research.

Objectives and Study Overview

This study aimed to develop and validate a high-throughput, HILIC-based LC-MS/MS method (LipidQuan) for simultaneous targeted screening of 24 ceramide species, 23 hexosylceramides, and sphingosine in human plasma. Key goals included minimizing isomeric interference, reducing the number of stable isotope-labeled standards, and achieving robust linear quantification over a wide dynamic range in an eight-minute run time.

Methodology

Sample preparation relied on simple protein precipitation with pre-cooled isopropanol (1:5 plasma:IPA) followed by centrifugation. Chromatographic separation was performed on an ACQUITY UPLC I-Class system using a BEH Amide column (2.1×100 mm, 1.7 μm) under HILIC conditions. A gradient from 0.1% to 20% mobile phase B over two minutes, then to 80% over three minutes, provided class-based elution: ceramides at ~0.4 min and hexosylceramides/sphingosine at ~0.8–1.1 min. Mass spectrometry employed positive-ion ESI MRM on Xevo TQ-S micro and TQ-XS instruments. Calibration curves used four deuterated ceramide standards spiked into plasma across relevant concentration ranges. Quantification acceptance criteria included R²>0.95 and CV<30% at the limit of quantification.

Instrumentation Used

• ACQUITY UPLC I-Class System
• BEH Amide 2.1×100 mm, 1.7 μm column
• Xevo TQ-S micro and Xevo TQ-XS triple quadrupole mass spectrometers
• Quanpedia method library
• TargetLynx and Skyline data processing software

Key Results and Discussion

The LipidQuan workflow achieved baseline separation of lipid classes, with ceramides eluting at ~0.4 min and hexosylceramides/sphingosine between 0.8 and 1.1 min, reducing isomeric/isobaric overlap. The method quantified 24 ceramide and 23 hexosylceramide species from 50 μL plasma with a linear dynamic range spanning four orders of magnitude. Representative calibration curves for C16, C18, and C24 ceramide-d7 standards exhibited R²>0.95 and acceptable CVs at LOQ. Utilizing class-specific surrogate standards lowered SIL requirements and overall assay costs while maintaining quantification accuracy.

Benefits and Practical Applications

This approach offers a rapid (<8 min) semi-quantitative platform for sphingolipid profiling suitable for large cohort studies, clinical screening, and comparative lipidomics. Key benefits include:

• High throughput and reduced run times
• Minimal sample volume and simple preparation
• Lower cost via fewer isotope-labeled standards
• Improved reproducibility across laboratories

Future Trends and Applications

Expanding the LipidQuan panel to additional sphingolipid subclasses and integrating orthogonal separation techniques (e.g., reversed-phase LC) could further enhance coverage. Automation of sample preparation and informatics pipelines, along with incorporation of more comprehensive isotopic standards, will support deeper mechanistic studies and clinical translation. Coupling targeted screens with non-targeted lipidomics may reveal novel biomarkers and therapeutic targets.

Conclusion

The HILIC-based LipidQuan LC-MS/MS method provides a fast, robust, and cost-effective solution for targeted ceramide and hexosylceramide screening in plasma. Its high sensitivity, broad dynamic range, and reduced isomeric interference make it well suited for biomarker research and routine clinical analysis.

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