Development and single-laboratory validation of an HPLC method for the determination of coumarin in foodstuffs using internal standardization and solid-phase extraction cleanup

A selective, sensitive and rapid procedure based on liquid chromatography with photodiode array detection (HPLC-PDA) has been developed, single laboratory validated and cross-validated by an external laboratory for the determination of coumarin, a naturally occurring biologically active principle, in a range of foods and beverages. Reverse phase solid-phase extraction (SPE) was used to clean sample extracts when co-extractives were found to interfere with detection and the presence of coumarin was confirmed by comparison of chromatographic peak UV spectra with coumarin standards and quantified by the use of an internal standard. The method was applied to the analysis of flour products, breakfast cereal, gelatin confectionery, sugar confectionery, rice pudding, mixed spice, camomile infusion and soft drinks. Coumarin could not be confirmed in extracts of camomile infusions due to the presence of co-eluting peaks as confirmed by UV spectral analysis. Chromatographic separation was achieved on an octadecyl (C8) column using a mobile phase gradient of acetonitrile and 0.5% acetic acid with a total run time of 20 minutes. The limits of detection and quantification were sample dependent and ranged from 0.05 to 2.5 mg kg(-1) and 0.05 to 8 mg kg(-1) respectively. The method was successfully validated to show the standard range linearity, sensitivity, accuracy and precision in the matrices tested. Coumarin is regulated within the European Union so this method may be readily adapted for enforcement purposes.