The aim of a recent study conducted by a team of Greek researchers (Kamaris et al., 2025) was to develop and validate a method based on the use of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the quantification of pyridoxine (PYR) and melatonin (MEL) in pharmaceutical chocolate formulations (for the treatment of insomnia).

The proposed method involves analysis on a reverse-phase column (250 mm × 4.6 mm, 5 µm) at 45 °C, using a mobile phase composed of two solvents (A, water with 0.2% formic acid; and B, acetonitrile with 0.2% formic acid) in a gradient elution at a flow rate of 1.2 mL/min. Sample preparation involves the following main steps: melting at 45 °C, solvent extraction (mixture of acetonitrile, methanol and formic acid), centrifugation, and final dilution of the supernatant with water.

The results show that the limits of detection for PYR and MEL are 0.09 and 0.92 ng/mL, respectively, while the limits of quantification are 0.29 and 3.04 ng/mL, respectively. Using this method, it was observed that 73.8% of PYR and 55.9% of MEL are released from the product and delivered to consumers’ intestines.

In conclusion, the authors argue that the proposed methodology can be usefully employed to develop new chocolate products with functional properties.

Bibliography: G. Kamaris et al., Chemistry, 7, 2025, 1-16.