Targeted LC/MS‐Based Quantitative Determination of 8 Endogenous Free Fatty Acids in Human Pleural Effusion Using Surrogate Analytes
Authors: Yip Lian Yee , Basri Nurhidayah , Lim Kah Ling , Tan Gek San , Tan Daniel Shao Weng , Lim Tony Kiat Hon , Ho Ying Swan 
Published: November 2023

Free fatty acids (FFA) such as oleic acid, docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) have recently been identified to be promising cancer biomarkers that can differentiate malignant from benign pleural effusion in the diagnosis of non‐small cell lung cancer (NSCLC). However, untargeted metabolomic approaches often used during early stages of biomarker discovery do not permit direct comparison of data measured across multiple platforms and experiments. This limitation poses a challenge for further validation of these FFA biomarkers for the diagnosis of NSCLC. Here, we developed and validated a targeted liquid chromatography‐mass spectrometry (LC/MS)‐based method using surrogate analytes to simultaneously quantify the absolute concentrations of a panel of 8 endogenous FFAs in human pleural effusion. These FFAs include 3‐hydroxypalmitic acid [FA16:0(3‐OH)], oleic acid (FA18:1), linoleic acid (FA18:2), arachidonic acid (FA20:4), eicosapentaenoic acid (EPA, FA20:5), docosatetraenoic acid (FA22:4), DPA (FA22:5) and DHA (FA22:6). FFAs in pleural effusion of benign and NSCLC patients were extracted using a modified Bligh and Dyer lipid extraction protocol. As the FFAs are present endogenously in the pleural effusion, calibration standards were constructed by spiking pleural effusion pooled from benign and NSCLC patients with serial dilutions of stable isotope labeled surrogate analytes. The concentrations of FFAs in the pleural effusion were back‐calculated based on the regression equation and response factor of the endogenous FFAs and their respective stable isotope labeled surrogate analytes. This methodology permitted preparation of calibration standards and quality control (QC) samples using pleural effusion as a matrix without interferences from the endogenous analytes. Lysophosphatidylcholine (LPC17:1) was used as the internal standard. Recovery, precision and accuracy of the method were assessed using QC samples (pooled pleural effusion) spiked with low, medium and high concentrations of surrogate analytes. Multiple reaction monitoring and single ion monitoring using electrospray ionization tandem mass spectrometry were used for the detection of all analytes. Our LC/MS/MS method enabled simultaneous quantification of 8 FFAs using an optimal selection of 5 FFAs surrogate analytes possessing the same carbon chain length of 18–22 carbon atoms as the endogenous FFAs. The linear range of the surrogate analytes were 0.6 – 26.1 μM for palmitoleic acid (U‐13C16), 0.2 – 65.8 μM for linoleic acid (U‐13C18) and oleic acid (U‐13C18), 0.2 – 44.1 μM for EPA‐d5 and DHA (U‐13C22). The recovery was consistent and precision and accuracy were within acceptable limits. A pilot study revealed oleic acid and linoleic acid were the predominant FFAs among the 8 FFAs in both benign (n=5) and malignant patients (n=5), and were 1.7‐fold higher in the malignant group. This LC/MS/MS method is precise, reliable and robust for the quantitation of FFAs in pleural effusion. It can be used to quantitate pleural effusion derived FFAs to evaluate their diagnostic performance as malignancy biomarkers for NSCLC.Support or Funding InformationThe financial support for this study is provided by the Biomedical Research Council of the Agency for Science, Technology and Research (A*STAR), Singapore, under the POLARIS program (SPF 2012/001).