Optimization of HPLC method for metanephrine and normetanephrine detection in urine: Enhancing diagnostic precision for pheochromocytoma

Catecholamines and their metabolites play critical physiological roles in the human body. Paragangliomas and pheochromocytomas are rare adrenal tumors that significantly alter catecholamine metabolism, particularly the concentrations of metanephrine (MN) and normetanephrine (NMN). This study presents the development and validation of a rapid and straightforward analytical method using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array (PDA) detector for quantifying MN and NMN in 24-h urine samples. Sample preparation involved adding 1 mL of urine to a tube containing the internal standard 3-methoxy-4-hydroxy benzylamine hydrochloride (MHBA) and a 2 g/L solution of 2-aminoethyl-diphenylborinate. After vortex mixing and centrifugation, ethyl acetate was used for extraction, and the organic layer was dried under nitrogen at 50–60 °C before reconstitution in the mobile phase. Chromatographic separation was achieved on an RP C-18 column with an isocratic flow of the mobile phase (sodium dihydrogen phosphate, citric acid monohydrate, acetonitrile, and sodium octyl sulfate). Detection was performed at 347 nm, with peak identification based on standard retention times. The method was validated for linearity (10–2000 ng/mL), recovery, sensitivity, precision, accuracy, selectivity, carryover, stability, and dilution effects. It showed a strong correlation coefficient (>0.99) and accuracy within ± 15 %. Inter- and intra-day precision confirmed the method reliability. This validated technique is suitable for clinical and research applications involving catecholamine metabolite screening.