A Validated Method for Quantification of Fatty Acids Incorporated in Human Plasma Phospholipids by Gas Chromatography-Triple Quadrupole Mass Spectrometry

Fatty acids (FA) are important mediators of health maintenance and disease risk. Optimal quantification assays of FA in high and low abundance as well the identification of C-13-labeled tracers to monitor FA metabolism are of major interest. The article on hand reports about the development and validation of a gas chromatography (GC)-triple quadrupole mass selective detection (GC-TQMS) method for absolute quantification of FA in human plasma phospholipids (hpPL). The quantification of the calibration solution by GC-flame ionization detection (GC-FID), with the introduction of a correction factor, allows the direct comparison of individual FA concentrations in hpPL by GC-TQMS. Specificity, sensitivity, and reproducibility are achieved by optimized chromatographic separation and employment of GC-TQMS. The inter-method comparison between GC-FID and GC-TQMS concentrations revealed good comparability for 27 FA. A full validation has been performed with linearity over 4 magnitudes, a limit of detection of 0.18-38 .3 fmol on column, a recovery of 83.6-109.6%, and intraday and interday precision data meeting the criteria of EMA and FDA guidelines. The method includes the absolute quantification of 58 positional and geometrical (cis/trans) isomeric FA in hpPL in the concentration range of 1-3000 nmol/mL, covering also low abundant positional cis/trans isomers. Results obtained from both methods are highly comparable, and selectivity and sensitivity are improved by using GC-TQMS. Additionally, we show here that calculation of C-13-labeled C16:0 tracer/tracee ratios in hpPL in human isotope enrichment studies is possible.

Automated summary

This study developed and validated a gas chromatography-triple quadrupole mass selective detection method for absolute quantification of fatty acids in human plasma phospholipids, allowing direct comparison of individual fatty acid concentrations and monitoring of C-13-labeled tracers. Results from both methods showed high comparability of FA concentrations and the method's accuracy and reproducibility were confirmed through full validation. Improved specificity and sensitivity were achieved using this GC-TQMS method, demonstrating its potential in human isotope enrichment studies.