Development and Validation of an HPLC-DAD Method for the Quantification of a Melatonin- Furanochalcone Hybrid: A Pilot Biodistribution Study and Potential Therapeutic Strategy Against Colorectal Cancer

Background: Colorectal cancer (CRC) remains a major cause of cancer-related mortality worldwide and ranks third in Colombia. Despite therapeutic advances, limitations such as reduced efficacy, adverse effects, and drug resistance persist. In the search for novel ther-apeutic strategies, molecular hybridization of melatonin and furanochalcone—compounds with antioxidant and antitumor properties—led to the synthesis of a novel hybrid mole-cule Mel-Fur (6f), a promising candidate for CRC treatment. Objectives: The aim of this study was to develop and validate an HPLC-DAD analytical method for quantification of Mel-Fur in serum and murine organ matrices. Methods: Chromatographic analysis used an Agilent Series 1200 system with a diode array detector and a C30 column under opti-mized conditions: acetonitrile: water (85:15, v/v) as mobile phase, flow rate 0.8 mL/min, detection at 342 nm, retention time 4.3 min. Results: The method fulfilled ICH Q2 (R1) and FDA validation guidelines, showing high selectivity, excellent linearity (R² > 0.999), sensitivity, precision, accuracy (RE% and CV% < 15%), recovery above 93%, and analyte stability for up to 8 days. The validated method was applied in a pilot in vivo biodistribu-tion study. Following oral administration of a single dose of Mel-Fur (1000 mg/kg) in BALB/c mice, rapid absorption and elimination were observed, with measurable systemic exposure and effective distribution into peripheral tissues. Pharmacokinetic analysis re-vealed preferential accumulation in lungs and liver, with sustained presence in colon. Conclusions: These findings provide a robust analytical tool and preliminary pharmaco-kinetic insights supporting further preclinical development of Mel-Fur as a potential therapeutic candidate for CRC.