Analytical Methods HPLC analysis and safety assessment of coumarin in foods Constanze Sproll, Winfried Ruge, Claudia Andlauer, Rolf Godelmann, Dirk W. Lachenmeier * Chemisches und Veterina ¨ runtersuchungsamt (CVUA) Karlsruhe, Weißenburger Str. 3, D-76187 Karlsruhe, Germany Received 11 April 2007; received in revised form 18 December 2007; accepted 27 December 2007 Abstract Coumarin is a component of natural flavourings including cassia, which is widely used in foods and pastries. The toxicity of coumarin has raised some concerns and food safety authorities have set a maximum limit of 2 mg/kg for foods and beverages in general, and a maximum level of 10 mg/l for alcoholic beverages. An efficient method for routine analysis of coumarin is liquid chromatography with diode array detection. The optimal sample preparation for foods containing cinnamon was investigated and found to be cold extraction of 15 g sample with 50 mL of methanol (80%, v/v) for 30 min using magnetic stirring. In the foods under investigation, appreciable amounts of coumarin were found in bakery products and breakfast cereals (mean 9 mg/ kg) with the highest concentrations up to 88 mg/kg in certain cookies flavoured with cinnamon. Other foods such as liqueurs, vodka, mulled wine, and milk products did not have coumarin concentrations above the maximum level. The safety assessment of coumarin containing foods, in the context of governmental food controls, is complicated as a toxicological basis for the maximum limits appears to be missing. The limits were derived at a time when a genotoxic mechanism was assumed. How- ever, this has since been disproven in more recent studies. Our exposure data on coumarin in bakery products show that there is still a need for a continued regulation of coumarin in foods. A toxicological re-evaluation of coumarin with the aim to derive scientifically founded maximum limits should be conducted with priority. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Cassia; Cinnamon; Coumarin; Flavourings; High-performance liquid chromatography (HPLC) 1. Introduction Coumarin is a natural substance occurring in the essen- tial oils of a number of plants used as flavouring ingredi- ents in foods. The occurrence of coumarin was reported in the following plant materials: Anthoxanthum odoratum (sweet vernal grass), Asperula odorata (sweet woodruff), Cinnamomum aromaticum (cassia bark), Dipterix odorata (tonka bean), Eupatorium triplinerve (white snakeroot), Hierochloe odorata (holy grass), Melilotus coerulea (sweet trefoil), M. officinalis (common melilot), Melittis melisso- phyllum (bastard balm), Primula elatior (oxlip) and Trilisa odoratissima (deer tongue) (MAFF, 1995). Coumarin’s aroma has been described as sweet, aromatic, a creamy vanilla bean odour with nut-like tones that are heavy, but not sharp or brilliant (Clark, 1995). Until 1954, when the first toxicological concerns about coumarin were raised, synthetic coumarin was widely used to add flavour, e.g. to the so-called maywines (second-grade white wine flavoured with woodruff) (Clark, 1995). After that, the use of couma- rin as a food flavouring was discontinued based on reports of hepatotoxicity prior to the existence of any carcinogenic- ity and mutagenicity data (Lake, 1999). According to Lake’s estimation, the main source of coumarin in the diet is cinnamon (Lake, 1999). A major source in alcoholic bev- erages is H. odorata, which is used to flavour a special kind of vodka, the so-called subrowka produced mainly in east- ern Europe (Nyka ¨nen, 1984). Coumarin was first suspected to have genotoxic and car- cinogenic effects in the 1980s (AFC, 2004). On this basis, 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.12.068 * Corresponding author. Tel.: +49 721 926 5434; fax: +49 721 926 5539. E-mail address: Lachenmeier@web.de (D.W. Lachenmeier). www.elsevier.com/locate/foodchem Available online at www.sciencedirect.com Food Chemistry 109 (2008) 462–469