Exploring chemical vs. enzymatic hydrolysis for fatty acid profiling of drying oils by liquid chromatography with high-resolution mass spectrometry

Drying oils, rich in triacylglycerols with unsaturated fatty acyl chains, are widely used in artworks for their ability to transform them into cross-linked polymer networks upon exposure to oxygen, forming protective layers. However, over time, ester bond hydrolysis releases fatty acids (FAs) that can aggregate, migrate, and interact with pigments, causing artwork degradation. Understanding the lipid profiles of siccative oils is critical for preserving cultural heritage. This study investigated the composition of free and total FAs in common drying oils, including safflower (Carthamus tinctorius), linseed (Linum usitatissimum L.), walnut (Juglans regia), and poppy seed (Papaver somniferum), using reversed-phase liquid chromatography (RPLC) coupled with electrospray ionisation (ESI) and high-resolution mass spectrometry (HRMS) and tandem HRMS (MS/MS). To determine the position and geometry (cis/trans) of CC double bonds in unsaturated FAs, a mild epoxidation reaction with meta-chloroperoxybenzoic acid was applied. Oils were analysed under three conditions: without prior hydrolysis, after enzymatic hydrolysis using Candida rugosa lipase (0.25 mg enzyme per mL of extracted sample, incubated at 37 °C for 45 minutes), or following chemical hydrolysis (0.5 M HCl, 100 °C, 1 h). The results demonstrate clear differences between hydrolysis methods: chemical hydrolysis induced isomerisation in the presence of metal traces, yielding additional unsaturated species compared to enzymatic hydrolysis. These transformations were characterised by MS/MS diagnostic ions of FA epoxides and their retention times. To preserve the native FA profile and minimise artefacts, enzymatic hydrolysis or mild chemical conditions are recommended, with C. rugosa lipase providing a reliable reference standard. This approach was successfully applied to a 1970s oil-on-canvas painting by F. Rispoli, revealing lipid alterations associated with aging.