Development of Profiling Method for Major Lipids in Blood by Triple Quadrupole LC/MS/MS

Importance of Topic

Lipid profiling in human blood is critical for understanding disease mechanisms, nutritional status, and metabolic health. Major lipid classes such as phospholipids, triacylglycerols, cholesteryl esters, free fatty acids, and cholesterol serve as biomarkers in clinical diagnostics and research on cardiovascular, inflammatory, and neurological disorders.

Objectives and Study Overview

This work aimed to expand an existing phospholipid profiling technique to include all major blood lipids. The goal was to develop a rapid, sensitive method capable of detecting over 100 lipid species from minimal sample volumes using triple quadrupole LC/MS/MS with polarity switching.

Methodology

Sample Preparation

• 5 µL human blood mixed with 500 µL methanol containing 0.1 % formic acid
• Centrifugation and direct use of 1 µL supernatant (equivalent to 10 nL blood) for analysis

Chromatographic Conditions

• UHPLC Column: Phenomenex Kinetex C8 (150 × 2.1 mm, 2.6 µm)
• Mobile Phase A: 20 mM ammonium formate in water; B: acetonitrile/isopropanol (50/50, v/v)
• Column Temperature: 50 °C; Flow Rate: 0.4 mL/min; Injection: 1 µL

Mass Spectrometry

• Instrument Mode: ESI with simultaneous positive/negative polarity switching (5 ms)
• Acquisition: Multi-reaction monitoring (MRM) with ~360 transitions
• Gas Flows: Nebulizing 3 L/min, Drying 10 L/min, Heating 10 L/min; Temperatures: DL 250 °C, Heater 400 °C, Interface 150 °C
• CID Gas Pressure: 270 kPa; Dwell/Pause: 3 ms/1 ms

Instrumentation

The analysis was performed on a Shimadzu Nexera UHPLC system coupled to a Shimadzu LCMS-8060 triple quadrupole mass spectrometer.

Results and Discussion

The method enabled identification of approximately 100 lipids from a 10 nL blood equivalent, including 51 phospholipids, 26 triacylglycerols, 11 cholesteryl esters, CE hydroperoxides, free fatty acids, and free cholesterol. The lipid class distribution in plasma was roughly 30 % phospholipids, 16 % triacylglycerols, 36 % cholesteryl esters, 14 % cholesterol, and 4 % free fatty acids. Detailed MRM chromatograms confirmed fatty acid combinations in 51 PLs and 26 TGs within a 20-minute run time.

Benefits and Practical Applications

• High sensitivity allows analysis from trace sample volumes
• Rapid throughput with 20-minute LC/MS/MS cycle
• Comprehensive coverage of major lipid classes in one assay
• Supports clinical biomarker discovery, nutritional studies, and lipidomic research

Future Trends and Opportunities

Advances may include integration with high-resolution mass spectrometry for isomer separation, automated sample preparation, expanded MRM libraries to cover oxidized lipids, and application to large-scale cohort studies. Data analysis pipelines using machine learning will further enhance biomarker identification and interpretation.

Conclusion

An MRM-based profiling method on the LCMS-8060 was successfully developed to detect over 100 major blood lipids from minimal sample volumes with high throughput and sensitivity. This approach enables detailed lipidomic analysis in clinical and research laboratories.

Reference

• Yamada M, Kubo H. Development of Profiling Method for Major Lipids in Blood by Triple Quadrupole LC/MS/MS. Shimadzu Application News No. C237, First Edition: Feb. 2021.