Development and validation of an NBD-F-based fluorometric method for the determination of plasma kynurenine and its clinical relevance in essential hypertension.

The kynurenine pathway, the main route of tryptophan catabolism, contributes to the pathogenesis of essential hypertension by promoting immune activation and endothelial dysfunction. Within cardiovascular disease, kynurenine (KYN) acts both as a diagnostic biomarker and as a mechanistic mediator. This study aimed to develop a sensitive fluorometric method for plasma KYN determination evaluating its clinical relevance in hypertensive cohorts. Because native KYN exhibits weak intrinsic fluorescence, derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was employed to generate a highly fluorescent derivative with enhanced spectroscopic properties, thereby improving the sensitivity of fluorometric detection. The resulting KYN-NBD derivative was measured at excitation and emission wavelengths of 485 and 555 nm, respectively. The reaction produced a stable KYN-NBD complex with a quantum yield of 0.63 compared with 0.22 for underivatized KYN, markedly improving method sensitivity. Validation according to ICH guidelines demonstrated linearity over 100-800 ng mL-1 (r 2 = 0.9996), high sensitivity (LOD: 10 ng mL; LOQ: 30 ng mL-1), and suitable accuracy and precision (recovery: 98.62-99.71%; RSD < 0.79%). The method additionally showed excellent selectivity without interference from confounding substances. To demonstrate its clinical applicability in a complex biological matrix, the validated assay was successfully applied to plasma samples obtained from 48 participants enrolled in a clinical case-control study, addressing the need for a simple and sensitive method suitable for routine plasma KYN determination. Plasma KYN levels were significantly higher in hypertensive individuals than in normotensive controls (510 ± 34 vs. 375 ± 25 ng mL-1, p < 0.001), correlating with systolic and diastolic blood pressure.