(T1330-01-02) MRM Based Free Fatty Acids Profiling in Human Plasma and Serum Using LC-MS/MS

Purpose: Free fatty acids, about 5% of the total fatty acids in the body, are known to be important signal transducers that regulate functions such as insulin secretion. It is also attracting attention as a biomarker for various endocrine disorders (1).Gas chromatography (GC) or gas chromatography-mass spectrometry (GC/MS) generally requires pretreatment such as methyl esterification for long-chain fatty acids and polyunsaturated fatty acids analysis. Multiple reaction monitoring (MRM) using liquid chromatography-mass spectrometry (LC/MS) enables highly sensitive quantitative analysis of fatty acids without derivatization. However, fatty acids widely found in the environment, such as palmitic acid and stearic acid, are introduced as impurities in LC system. It has been reported that there is difficulty in accurate quantification of these fatty acids in real samples analysis (2). This study presents an example of quantitative analysis of free fatty acids in human plasma and serum using LC-MS/MS coupled with a delay column.  Through the use of the delay column, system-derived contaminants are time-separated from sample-derived fatty acids allowing accurate quantitation without complex mitigation efforts.

Methods: Chemical standards were purchased from Cayman Chemical (Ann Arbor, MI). Human plasma from human volunteer obtained from Kojin-Bio Co. (Japan). Two types of human plasma were used: EDTA plasma and heparin plasma. 295 μL of acetonitrile containing 1% formic acid and 5 μL of internal standard were added to 100 μL of plasma and serum followed by stirring for about 1 minute. After centrifugation, the supernatant was collected as it was used as an analytical sample. They were diluted with acetonitrile containing 1% formic acid and subjected to LC/MS/MS analysis. The internal standard added was adjusted to a final concentration of 25 ng/mL according to the dilution ratio. An UHPLC system (Shimadzu Nexera) and triple quadrupole mass spectrometer (Shimadzu LCMS-8060RX) were used. The analytical conditions for HPLC as well as MS are shown in table format.  A delay column has been installed between the gradient mixer and the autosampler to improve the separation of target and environmental contaminant components from mobile phase. A simultaneous analytical method was developed for the following compounds: Lauric Acid, Stearidonic Acid, Eicosapentaenoic Acid (EPA), α- Linolenic Acid, γ-Linolenic Acid, Myristic Acid, Docosahexaenoic Acid (DHA), Palmitoleic Acid, Arachidonic Acid (AA), Linoleic Acid, Docosapentaenoic Acid (DPA), Dihomo-γ-Linolenic Acid (DGLA), Palmitic Acid, Adrenic Acid, Oleic Acid, Stearic Acid, Arachidic Acid, Nervonic Acid, and Lignoceric Acid.

Results: By installing a delay column between the mixer and the autosampler, target compounds derived from the sample were able to be separated from contaminants in the mobile phase. The wide broad peaks, which are considered to be fatty acids derived from the mobile phase, were detected after the peaks of several free fatty acids (palmitic acid C16:0, stearic acid C18:0 and oleic acid C18:1) in the plasma and serum samples. Without the delay column, the peak from the blood compound overlapped with the broad peak from the mobile phase, and the accurate peak area could not be obtained. Internal standard method was used for the calibration curve in each compound, with three or more calibration points in the range of 1-1000 ng/mL. We confirmed that good linearity with the contribution ratio (R²) >0.999 was obtained for all compounds. Good results were obtained for all compounds, with an accuracy of 70-130% of the mean concentration and repeatability (%RSD) of less than 10% at the lowest calibration point. Pretreated human plasma and human serum were diluted with acetonitrile containing 1% formic acid according to the content and subjected to quantitative analysis. The results of conversion to the concentration in the undiluted solution are summarized in table format. All 19 compounds were detected, and good repeatability of the estimated concentrations with RSD < 10% was obtained.

Conclusion: Simultaneous analysis of 19 free fatty acids in human plasma and serum was performed using UHPLC-MS/MS without interference from environmental sources of common fatty acids. Good linearity was obtained in the range of 1-1000 for all compounds, and we also confirmed that the repeatability at lowest calibration point was less than 10%. The use of a delay column allowed this analytical method to provide more stable and accurate quantitative analysis with simple pretreatment.

References: (1) Takahashi H, et al., “Long-Chain Free Fatty Acid Profiling Analysis by Liquid Chromatography–Mass Spectrometry in Mouse Treated with Peroxisome Proliferator-Activated Receptor α Agonist”, Biosci. Biotechnol. Biochem., 77, 2288–2293 (2013).
(2) Okada H., et al., “Development of a liquid chromatography–electrospray ionization tandem mass spectrometric method for the simultaneous analysis of free fatty acids”, J. Biochem., 170(3):389-397 (2021).


Analysis Conditions


MRM Chromatograms of 19 free fatty acids, note broad peaks generated by delay column versus sample-derived peaks that elute prior.


Concentration of 19 free fatty acids in human plasma and serum