Florescence based analytical assessment of plasma neopterin and its clinical relevance in hypertension.

Neopterin (NPN), a pteridine derivative produced by activated monocytes and macrophages in response to interferon-γ, is a recognized biomarker of cellular immune activation, with elevated plasma levels reported in infections, autoimmune diseases, cardiovascular disorders, and cancers. There is an increasing attention given to its role in oxidative stress and vascular dysfunction in hypertension. In this work, a fluorescence-based method was developed and validated for the determination of NPN in plasma using derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The reaction produced a stable fluorescent conjugate with a quantum yield of 0.61 compared to 0.21 for native NPN, resulting in enhanced signal intensity and improved selectivity. The method showed linearity over the concentration range of 1–20 ng/mL with a correlation coefficient of 0.9997. The limits of detection and quantification were 0.15 ng/mL and 0.45 ng/mL, respectively, with a mean recovery of 98.54%. Intra-day and inter-day relative standard deviations were below 2.5%, and interference from structurally related compounds was negligible (< 4%). The validated method was applied in a clinical case–control study of 52 participants (26 hypertensive patients and 26 normotensive controls), revealing significantly higher plasma NPN levels in hypertensive patients (3.0 ± 0.7 ng/mL) compared to controls (1.8 ± 0.4 ng/mL, p = 0.0002), with positive correlations to systolic (r = 0.48, p = 0.009) and diastolic blood pressure (r = 0.42, p = 0.025). These findings demonstrate that the developed method provides a sensitive and reliable approach for plasma NPN quantification and support its potential as a biomarker of hypertension diseases.