Comprehensive Analysis of Different Subtypes of Oxylipins to Determine a LC–MS/MS Approach in Clinical Research

Figures

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  Figure 1. LA-, DGLA-, AA-, ALA-, EPA-, and DHA-derived oxylipins and the main enzymes involved in their generation and breakdown. Arrows of different colors represent distinct pathways.

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  Figure 2. Evaluation of five oxylipin extraction methods based on feature detection and metabolic profiling. (A) Bar graph showing the count of consistently detected oxylipins for each method. mSPE and PPT methods (MeOH, ACN) detected more features than LLE methods (MTBE, EA). (B) PCA score plot illustrating the metabolic variation introduced by different extraction protocols. The tight cluster of SQC samples confirms system stability. Distinct, method-dependent clustering is observed: PPT methods (MeOH, ACN) and LLE methods (MTBE, EA) form two separate, tight clusters, while the mSPE protocol separates from both and shows greater dispersion among its replicates.

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  Figure 3. Comparison of extraction efficiency across different protocols. (A) Radar diagrams of peak area of IS extracted from different protocols MeOH, ACN, EA, MTBE, and mSPE. For each extraction method n = 3. (B) Heatmaps reflecting the peak area of standard unlabeled analytes from serum. “1” represents the maximum peak area in five protocols; “0” represents the minimum peak area in five protocols.

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  Figure 4. Summary of extraction recoveries obtained for standards spiked (25 ng/mL) in serum samples for each sample preparation protocol. (A) Comparison of extraction recoveries using different extraction methods. (B) Comparison of extraction recoveries of nine ISs using different extraction methods. (C) The extraction recovery rate of different methods ranges from 80% to 120%.

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  Figure 5. Comparison of mSPE and aSPE. (A) Histograms of detected features in each protocol are used to assess the extraction efficiency. (B) The coefficient of variation (CV%) of peak intensity for 50 compounds detected by both manual and aSPE. (C) Extraction recoveries with mSPE and aSPE. (D) Matrix pie chart of analytes attributed to various MEs.

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  Figure 6. Comparison of the extraction recoveries obtained for 50 standards and MEs obtained for standards spiked (25 ng/mL) in serum samples with sample reconstituted by MeOH or 50% ACN (n = 3 for each reconstitution method).

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  Figure 7. Serum oxylipins concentrations in the CHD and healthy control groups, as measured by UPLC–MS/MS. Data are means ± SEM. Results were compared using Student’s t-tests. n = 30–31 per group. * p < 0.05 and *** p < 0.001 vs. CHD group. p-value corrected by Bonferroni.

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  Figure 8. ROC curves of the 11-HETE, 15-HETE, 5-HETE, and the integrated biomarker for predicting the CHD.