Ultrasensitive detection of neurotransmitter dopamine in human urine via fluorescent azamonardine formed with resorcinol

This work presents a pre-column derivatization HPLC–spectrofluorimetric platform for the ultratrace quantification of endogenous dopamine (DA) in human urine. Under alkaline conditions, DA rapidly condenses with resorcinol to form a highly fluorescent azamonardine derivative ( λ ex / λ em = 420/460 nm), whose emission properties provide excellent analytical sensitivity in complex biochemical matrices. The derivatization proceeds quickly and yields a product with fluorescence stability for up to 6 hours. Key derivatization parameters were optimized using a face-centered central composite design. Selectivity studies confirmed the method’s discriminative capability against major metabolic interferents (e.g., homovanillic acid), enabling accurate quantitation at low-ppb concentrations. Method validation was performed using accuracy profiles in accordance with FDA bioanalytical guidelines. The method exhibited linearity over 5–100 ng/mL with a limit of detection of 0.3 ng/mL. The method precision was less 3.5% in all cases. Environmental and operational performance were simultaneously assessed using the unified multicolor MA tool, demonstrating the method’s strong sustainability profile and practical feasibility. Application to authentic human urine samples further confirmed the system’s translational potential as a robust, high-performance analytical approach for clinical diagnostics, neurochemical studies, and broader bioanalytical applications.