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

[Image: see text] 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 (13)C-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 (13)C-labeled C16:0 tracer/tracee ratios in hpPL in human isotope enrichment studies is possible.