Development of an MRM based phospholipid profiling method in human plasma using an inert C18 column

Significance of the topic

Phospholipids in human plasma play critical roles in cellular function and are promising biomarkers for disease and pathophysiological research. A robust, high‐throughput profiling method enables comprehensive lipidome analysis, supporting biomarker discovery and metabolic studies.

Objectives and overview of the study

This work aimed to develop and validate a rapid, sensitive multiple reaction monitoring (MRM) LC–MS/MS method for profiling phospholipids in human plasma. Key goals included:

• Establishing a 20-minute chromatography cycle to detect a wide range of phospholipid classes.
• Optimizing MRM transitions for both polar head groups and fatty acid fragments.
• Evaluating reproducibility, coverage, and carryover.

Methods and instrumentation

Sample preparation involved protein precipitation of 20 µL plasma with 1 mL methanol containing 0.1% formic acid, followed by centrifugation and 3 µL injection.

• Liquid chromatography: Shim-pack Scepter Claris C18 column (2.1 × 100 mm, 1.9 µm) at 50 °C, flow rate 0.3 mL/min; curved gradient using 20 mM ammonium formate (A) and acetonitrile/isopropanol (1:1, v/v) (B).
• Mass spectrometry: Nexera UHPLC coupled to LCMS-8060NX triple quadrupole with ultra-fast polarity switching (5 ms), ESI in positive/negative modes, DL temp. 250 °C, block heater 400 °C, interface 150 °C, CID gas 270 kPa.
• MRM setup: 662 transitions covering polar head group fragment m/z 184 for phosphatidylcholine derivatives and one–two transitions per diacyl species in negative mode to assign fatty acid composition.

Main results and discussion

The method achieved comprehensive coverage and high reproducibility:

• Detected 170 phospholipids within a 20-minute run, including 130 diacyl-phospholipids, 28 lyso-phospholipids, and 12 sphingomyelins.
• Reproducibility assessment over 50 injections: 107 compounds exhibited CV ≤ 20% and all 170 compounds CV ≤ 50%.
• Carryover below 0.01% for the most abundant PC 16:0_18:2 peak.
• Curved gradient and inert C18 column enabled partial separation of isomeric species such as PC 18:2_20:4, 18:1_20:5, and 16:0_22:6, with distinct retention times.
• Clear detection of omega-3 fatty acid–containing species (EPA, DHA) bound to phospholipids supports fatty acid balance evaluation.

Benefits and practical applications of the method

This workflow provides:

• High‐throughput, reproducible phospholipid profiling suitable for large‐scale biomarker discovery studies.
• Quantitative assessment of fatty acid composition in phospholipid classes for metabolic and nutritional research.
• Potential application in clinical and quality‐control laboratories for monitoring lipid alterations in disease states.

Future trends and potential applications

Advancements may include integration with high‐resolution MS for structural elucidation, automation of sample processing, and expansion to other biofluids. Coupling this approach with lipidomics data analytics and machine learning could accelerate biomarker identification and personalized nutrition studies.

Conclusion

An MRM-based LC–MS/MS method using an inert C18 column was successfully developed for rapid, comprehensive phospholipid profiling in human plasma. The protocol delivers broad coverage, strong reproducibility, minimal carryover, and the ability to resolve key isomers, positioning it as a valuable tool in clinical lipidomics and biomarker research.