Determination of red blood cell fatty acid profiles in clinical research

Importance of the Topic

Fatty acid composition in red blood cells (RBCs) is recognized as a valuable biomarker in various human diseases. Traditional methods based on gas chromatography with flame ionization or electron impact mass spectrometry require long run times and extensive separation to achieve confident identification and quantification. A more rapid, sensitive, and robust method is essential for high-throughput clinical research applications.

Objectives and Study Overview

This study aimed to develop and validate a fast and reliable analytical workflow for profiling RBC fatty acids. Key goals included:

• Derivatization of fatty acids to methyl esters (FAMEs).
• Implementation of ammonia-induced chemical ionization (CI) with gas chromatography tandem mass spectrometry (GC/MS/MS).
• Comparison against conventional single quadrupole GC/MS with electron impact (EI).
• Application to over 700 clinical RBC samples to demonstrate throughput and performance.

Methodology and Sample Preparation

Red blood cells were washed with saline, hemolyzed in water, and centrifuged. Fatty acids were extracted, spiked with a heptadecenoate internal standard, and dried under nitrogen. Derivatization to FAMEs was performed using boron trifluoride in methanol at 100 °C. After phase separation with hexane and water, samples were evaporated and reconstituted in hexane before GC/MS/MS analysis.

Used Instrumentation

• Agilent 7890A GC with split/splitless inlet coupled to Agilent 5975C MS (EI) for method comparison.
• Agilent 7890A GC with Agilent 7000 MS/MS (CI) using ammonia as reagent gas.
• Agilent J&W CP-Sil 88 FAME columns (100 m × 0.25 mm, 0.20 µm) and a 5 m uncoated retention gap.
• Carrier gas: helium at 2.0–2.2 mL/min; CI reagent gas: ammonia at 1.3 mL/min.
• MRM acquisition mode targeting molecular ions [M+H]+ and ammonia adducts [M+NH4]+.

Main Results and Discussion

The CI-GC/MS/MS approach delivered dominant molecular ion signals for FAMEs, overcoming the extensive fragmentation seen in EI and enabling compound-specific quantification. Key findings included:

• Analysis time reduced to nine minutes versus up to 60 minutes for EI-GC/MS.
• Calibration linearity with R2 > 0.995 in solvent and > 0.992 in matrix across 45 FAMEs.
• Limits of detection and quantification in the low ng/mL range.
• Intra- and inter-assay precision (RSD) below 10 % for all monitored fatty acids.
• Clear chromatographic separation with no observed interferences, even for coeluting isomers.

Benefits and Practical Applications

By combining ammonia CI with tandem mass spectrometry, the method offers:

• High-throughput capability for large clinical studies.
• Enhanced sensitivity and selectivity through molecular ion monitoring.
• Reliable quantification of 45 fatty acids in RBCs.
• Reduced solvent and instrument time with robust performance.

Future Trends and Possibilities of Use

Potential developments include:

• Automation of sample preparation and data processing for further throughput gains.
• Extension to other lipid classes or biological matrices such as plasma or tissues.
• Exploration of alternative CI reagents or column chemistries to further shorten run times.
• Application in large-scale epidemiological or nutritional intervention studies.

Conclusion

The developed ammonia-CI GC/MS/MS method enables rapid, sensitive, and accurate profiling of red blood cell fatty acids. Its short run time, high selectivity, and reproducibility make it well suited for high-throughput clinical research, overcoming limitations of conventional EI-based assays.

References

1. Boecking C et al. Development and validation of a combined method for the biomonitoring of omega-3/-6 fatty acids and conjugated linoleic acids in different matrices from human and nutritional sources. Clin Chem Lab Med. 2010;48:1757–1763.
2. Dodds ED et al. Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact mass spectrometry. Lipids. 2005;40:419–428.