Development and validation of an HPLC method to determine metabolites of 5‐hydroxymethylfurfural (5‐ HMF )

The food component 5‐hydroxymethylfurfural is supposed to have antioxidative properties and is therefore used as an acting agent in a novel anticancer infusion solution, named K aral®, and an oral supplementation. Previous studies showed that after oral and intravenous application, the substance is completely decomposed to its metabolites: 5‐hydroxymethylfuroic acid, 2,5‐furandicarboxylic acid, and N ‐(hydroxymethyl)furoyl glycine. The formation of a fourth metabolite, namely 5‐sulphoxymethylfurfural, is still not clarified according to literature. Due to commercial unavailability, synthesis of 5‐sulphoxymethylfurfural was conducted and a synthesis procedure for N ‐(hydroxymethyl)furoyl glycine had to be developed. Identification of the synthesised compounds was proven by LC ‐ MS and NMR . An appropriate HPLC method was established to obtain good separation of the four possible metabolic substances and 5‐hydroxymethylfurfural within 12 min via a HILIC column (150 × 4.6 mm, 5 μm) using a gradient grade system switching from mobile phase A ( ACN /ammonium formate 100 mM, p H 2.35, 95:5, v/v) to mobile phase B ( ACN /ammonium formate 100 mM, p H 2.35, 85:15, v/v). The procedure was afterward validated following ICH guidelines in terms of selectivity, linearity, precision, LOD , and LOQ .