Targeted metabolomics for cardiovascular disease: Validation of a high-throughput HPLC-MS/MS assay

Cardiovascular diseases (CVD) remain a leading cause of mortality worldwide, necessitating innovative diagnostic tools to improve early detection and management. This study represents the optimization and validation of a high-throughput HPLC-MS/MS method for simultaneous quantification of 98 metabolites in human plasma, including amino acids and its derivatives (n = 29), tryptophan pathway metabolites (n = 17), nucleosides (n = 4), water-soluble vitamins (n = 3), acylcarnitines (n = 39), and others (n = 6). The method utilizes chemical derivatization to enhance retention and sensitivity of polar metabolites providing accurate analysis across diverse physicochemical properties. The presented method was validated in accordance with EMA guidelines and included assessment of linearity, accuracy, precision, matrix effects, recovery, and stability. Parallelism testing confirmed the suitability of a surrogate matrix for calibration. The method was applied for the analysis of plasma samples from 399 patients with cardiovascular diseases and 75 healthy controls, revealing significant metabolic alterations in pathways associated with inflammation, nitric oxide metabolism, and mitochondrial function. The presented comprehensive approach may serve as a rapid screening method for the identification of selective CVD biomarkers using targeted metabolomic profiling.