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

Abstract

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 ¹³C-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 ¹³C-labeled C16:0 tracer/tracee ratios in hpPL in human isotope enrichment studies is possible.

Figure 1

Concentrations (in nmol/mL) of 49 saturated and unsaturated cis/trans isomeric fatty acids in the range from C14 to C26 chain length. Box-plot parameters show the mean and the standard deviation of the fatty acid concentrations quantified in human phospholipids in 37 samples obtained from healthy volunteers. Phospholipids derived from human plasma were enriched by solid phase extraction on an amino propyl column. Fatty acids in enriched human phospholipid fraction were derivatized to fatty acid methyl ethers (FAMEs) prior to chromatographic separation on an Agilent 7890A gas chromatograph equipped with a CP-Sil88 column (100 m in length, 0.25 mm in diameter, and <0.192 μm film thickness, Agilent J&W). Fatty acid methyl esters were detected in positive multiple reaction monitoring mode (MRM) by quantifier and identifier transitions for saturated (143.1 → 55.1 m/z; 143 → 101.1 m/z), mono-unsaturated (97.1 → 55.1 m/z; 97.1 → 69.1 m/z), and poly unsaturated (79.1 → 77.1 m/z; 79.1 → 51.1 m/z) fatty acid methyl esters. The total runtime was 85 min, and FAMEs were quantified against an external calibration curve on an Agilent 7000 TQ-MS. As internal standard, a D₃₅-C18:0 FAME was used.

Figure 2

Retention time and limits of detection and quantification. (A) Retention time of included fatty acids ordered by chain length and saturation. (B) Limit of detection and quantification ordered by decreasing sensitivity.

Figure 3

Bland–Altman plots and correlations for the most abundant saturated and ω-3, ω-6, and ω-9 fatty acids in (A) to (D), respectively.