Targeted ultra-high performance liquid chromatography-tandem mass spectrometry assay for the quantification of medium-chain phosphatidylcholines in platelets of coronary artery disease patients

In a recent untargeted clinical lipidomics study of platelets of coronary artery disease (CAD) patients, medium-chain phosphatidylcholines (MCPCs) with C8 and C10 fatty acyl residues were found significantly upregulated in the patient group with acute coronary syndrome (ACS) as compared to chronic coronary syndrome (CCS) and healthy controls. To support this finding, this work presents the development and optimization of a targeted UHPLC-QTrap-MS/MS method with multiple reaction monitoring acquisition for the quantitative analysis of MCPCs (PC 10:0/8:0, PC 16:0/8:0, PC 10:0/20:4 and PC 10:0/10:0) in platelets for biomarker validation. A systematic optimization of chromatographic and mass spectrometric parameters was performed. A charged surface hybrid CSH C18 (1.7 µm, 130 Å) column and fine-tuned gradient elution with 2-propanol/acetonitrile and ammonium acetate as additive to the mobile phase was employed in the final method in ESI negative mode. Four selected PC standards (PC 6:0/6:0, PC 8:0/8:0, PC 10:0/10:0 and PC 12:0/12:0), which cover well the carbon and retention rime range of the target analytes, were used for the optimization process and calibration. Quantification was based on matrix-matched calibration with these four selected commercially available MCPC standards as surrogate calibrants and PC 6:0/6:0(d22) as internal standard. Furthermore, an organic solvent and fatty acyl carbon number-corrected response factor approach gave also accuracies within acceptance limits of bioanalytical validation guidelines and has more generic applicability. Compared to the previous untargeted RPLC-ESI-QTOF-MS/MS method, the optimized targeted UHPLC-QTrap-MS/MS assay showed increased sensitivity and selectivity for the detection of medium-chain PCs in platelet samples of CAD (LOQs in the range of 0.5–5 nmol/L). The method performance parameters indicated its suitability for a future biomarker validation study of MCPCs in platelets.