Advancing Targeted Metabolomics Using Cyanopropyl-Based Liquid Chromatography Tandem Mass Spectrometry

Abstract

The change of metabolic pathways is recognized as the key to disease discovery prompting the development of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based quantitative platforms to explore the dynamic metabolite profiles of organisms. In this study, a liquid chromatography method based on cyanopropyl (CN) was developed. By adjusting the pH environment of the column, we achieved the elution of 51 metabolites spanning the most comprehensive set of biological pathways currently known. Offering rapid chromatography, efficient separation, and green chemistry benefits, the method encompasses nucleosides and nucleotides, the oxidative-redox metabolome, the glycolysis pathway, the pentose phosphate pathway, the purine de novo pathway, amino acids, and neurological disorder-related metabolites. The mass spectrometry was equipped with electrospray ionization in both positive and negative modes with scheduled multiple reactions monitoring. The validation of the method involved a comprehensive assessment of linearity, accuracy, precision, and matrix effect. The linear range was from 1.0 to 2000 ng mL^–1 with a high correlation coefficient (r > 0.99). The LOD ranged from 0.1 to 10 ng mL^–1, and the LOQ ranged from 0.1 to 25 ng mL^–1. The overall recovery ranged from 81.3% to 117.8%, with RSD < 15.1%. Subsequently, an analysis of metabolites was conducted in dSH-SY5Y neuroblastoma cells with 6-hydroxydopamine, a commonly used neurotoxin in neurodegenerative diseases. The results demonstrate that neurotoxin-induced mitochondrial damage significantly altered related analytes, corroborating previous estimates and validating the feasibility and reliability of the bioanalytical platform.