Development of an Analytical Method for Blood Triglycerides Using Triple Quadrupole Mass Spectrometer

Significance of the Topic

Blood triglycerides represent a critical class of lipids involved in energy storage and transport. Elevated levels are associated with cardiovascular and metabolic disorders. Detailed profiling of individual triglyceride (TG) species and their fatty acid composition provides deeper insight into disease mechanisms and biomarker discovery.

Objectives and Overview of the Study

The authors aimed to develop a high-throughput LC-MS/MS method for simultaneous quantification of 47 TG species in human blood within 11 minutes. The approach incorporates multiple reaction monitoring (MRM) transitions to characterize fatty acid composition bound to glycerol, enhancing conventional total TG assays.

Methodology and Instrumentation

A Shimadzu Nexera UHPLC system coupled to the LCMS-8060 triple quadrupole mass spectrometer was employed. Chromatographic separation utilized a C18 column (Shim-pack Velox™, 50×2.1 mm, 2.7 µm) at 45 °C. Mobile phases were 20 mM ammonium formate in water (A) and 2-propanol/acetonitrile (80/20, v/v; B) delivered at 0.4 mL/min. A 3 µL injection volume enabled 130 analyses per day.

Mass spectrometry operated in positive electrospray ionization with MRM mode. Ammonium adducts served as precursor ions, and neutral loss of fatty acids (C14–C22, unsaturation 0–6) defined product ions. Gas flows, temperatures, and pressures were optimized for sensitivity. Glyceryl trilinolenate (TG 54:9) quantified as internal standard.

Sample preparation involved protein precipitation of 20 µL plasma or serum with methanol/butanol (1:1) and internal standard, followed by stirring, centrifugation, dilution, and LC-MS/MS injection in triplicate.

Main Results and Discussion

The MRM library comprising 195 transitions captured 47 TG species and enabled detection of isomeric fatty acid combinations. Representative chromatograms showed TG 52:2 as the most abundant species in plasma. Neutral loss monitoring identified polyunsaturated and odd-chain fatty acids. A total of 181 components with reproducibility (RSD ≤15%) were retained for data analysis.

Multivariate analysis using principal component analysis (PCA) clearly separated two plasma samples and one serum sample into distinct clusters. One-way ANOVA revealed significant differences (p<0.01) for 109 TG components. Comparative bar plots highlighted TG species preferentially enriched in each sample type, demonstrating the method’s ability to resolve subtle lipidomic variations.

Benefits and Practical Applications

• Rapid quantification of diverse TG species in under 11 minutes per run.
• High throughput supports large-scale biomarker screening (≈130 analyses/day).
• Detailed fatty acid profiling informs metabolic and clinical research.

Future Trends and Applications

Advancements may include expanding MRM libraries to cover broader lipid classes, integration with high-resolution MS for structural elucidation, and automation of sample preparation. Coupling lipid profiling with clinical cohorts could accelerate biomarker discovery and personalized medicine applications.

Conclusion

The developed LC-MS/MS MRM method offers a robust, high-throughput platform for comprehensive triglyceride profiling in human blood. Its sensitivity and specificity for individual TG species and fatty acid compositions make it a valuable tool for biomarker research and lipidomics studies.

References

1. Liebisch G, Fahy E, Aoki J, et al. Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures. J Lipid Res. 2020;61(12):1539-1555. doi:10.1194/jlr.S120001025