A rapid LC-MS/MS method for the simultaneous quantification of 10 key folate cycle intermediates in human plasma.

The folate cycle is essential for regulating metabolic processes. Simultaneous measurement of folate cycle intermediates is crucial for understanding metabolic disruptions in hematopoietic, nervous, renal and cardiovascular diseases. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for folate cycle metabolites showed poor retention for highly polar compounds in reversed-phase separations with long analytical run time (30 min). Herein, we developed a novel LC-MS/MS method using hydrophilic interaction liquid chromatography (HILIC) mode for simultaneous measurement of 10 key folate cycle metabolites in human plasma, including 5 folate intermediates, 4 related amino acids, and a cofactor (VB₁₂), with enhanced chromatographic retention and reduced analysis time (8.5 min) without derivatization. Through further method validation, all analytes demonstrated acceptable linearity (R² > 0.989), precision (intra-day precision: 1.3-11.3 %; inter-day precision: 3.4-14.6 %), recovery (89.5-113.8 %) and reasonable matrix effect (81.6-115.8 %). The results presented that all intermediates were stable for 5 h at 5°C (autosampler), 12 h at -40°C and 24 h at -80°C. Moreover, the method was successfully applied in clinical plasma from critically ill patients, revealing distinct metabolic perturbations in acute kidney injury (AKI) inpatients compared with non-AKI controls (NAKI). Levels of 5-MTHF and Gly were significantly elevated in the AKI group. Correlation analysis revealed that SCr levels were positively correlated with both 5-MTHF (r = 0.26, p = 0.04) and hCys (r = 0.27, p = 0.04) concentrations. The study is promising to evaluate folate nutritional status to mitigate the risks of folate-related diseases, such as megaloblastic anemia and neural tube defects.