Direct infusion nano-electrospray ionization mass spectrometry for untargeted metabolomics of trace-volume aqueous humor from prematurity infants.

Reactivation of retinopathy of prematurity (ROP) after anti-VEGF therapy poses a serious risk of retinal detachment. Metabolomics of aqueous humor (AH) holds promise for the discovery of early biomarkers, but the extremely limited sample volumes (as low as 2 μL) from prematurity infants hinders traditional liquid chromatography-mass spectrometry (LC-MS). Here, we applied direct infusion nano-electrospray ionization mass spectrometry (DI-nESI-MS) for metabolomic profiling of trace-volume AH samples without chromatographic separation. By comparing the aqueous humor of infants with and without ROP reactivation, as well as before and after treatment, we found significant metabolic changes, including abnormal fatty acid oxidation, inhibition of glycolysis and dysregulation of the serine-glycine pathway. Elevated ascorbic acid levels are regarded as a potential protective factor. Our research established DI-nESI-MS as a powerful tool for the analysis of extremely small amounts of biological fluids and revealed new metabolic insights into ROP reactivation.