Comprehensive quantification of C4 to C26 free fatty acids using a supercritical fluid chromatography-mass spectrometry method in pharmaceutical-grade egg yolk powders intended for total parenteral nutrition use.

Free fatty acids (FFAs) are important energy sources and significant for energy transport in the body. They also play a crucial role in cellular oxidative stress responses, following cell membrane depolarization, making accurate quantification of FFAs essential. This study presents a novel supercritical fluid chromatography-mass spectrometry (SFC-MS) method using selected ion recording in negative electrospray ionization mode, enabling rapid quantification of 31 FFAs within 6 min without derivatization. FFAs are identified and quantified using an HSS C18 SB column and a secondary mobile phase consisting of methanol with formic acid by detecting their [M - H] ions. Calibration curves showed strong linearity (R ≥ 0.9910), spanning 1000-12,000 ng/mL for short-chain FFAs and 50-1200 ng/mL for medium- and long-chain FFAs. The method achieves detection limits as low as 1 ng/µL for short-chain FFAs and 0.05 pg/µL for other FFAs per on-column injection. The method demonstrated high accuracy and precision, with bias and coefficients of variation maintained below 15% across five quality control levels. Freeze-thaw and autosampler stability studies confirmed the behavior of matrix-matched standards under optimal storage conditions. The validated method was applied to the analysis of pharmaceutical-grade egg yolk powders, using 13 deuterated FFAs as internal standards (IS) in comparison with heptadecanoic acid (C17:0). Significant variations in FFA quantification using two different IS approaches underscore the importance of selecting an appropriate IS. In summary, this study introduces a reliable and validated SFC-MS method for analyzing FFAs ranging from C4 to C26, requiring minimal sample preparation.