FOOD COMPOSITION AND ADDITIVES Single Laboratory Validation of a Method for the Determination of Hydroxymethylfurfural in Honey by Using Solid-Phase Extraction Cleanup and Liquid Chromatography MALCOLM DRIFFIELD,DANNY CHAN,ROY MACARTHUR,SUSAN MACDONALD, and PAUL BRERETON Central Science Laboratory, Sand Hutton, York, YO41 1LZ, United Kingdom ROGER WOOD Food Standards Agency, Aviation House, 125 Kingsway, London, WC2B 6NH, United Kingdom A method is described for the determination of hydroxymethylfurfural (HMF) in honey. The method, which is based on solid-phase extraction cleanup followed by liquid chromatography (LC) with UV absorbance detection, was tested on a variety of different honey types: liquid, set, blended, filtered, crystalline, and comb honey. A sample of honey fortified with a known amount of HMF acted as an in-house reference material. LC with diode-array detection showed that the HMF peak did not contain any peaks of coeluting interfering species. Stability studies showed that honey samples should not be repeatedly frozen and thawed because the temperature changes caused a gradual increase in the HMF concentration. It was also shown that aqueous HMF standard solutions should be kept in the dark at 4°C to avoid degradation of the HMF. The method was internally validated, and the measurement uncertainty was estimated to be ±9.0 at 40 mg/kg, the legal limit. A comparison of the relative standard uncertainty with the Horwitz relative standard deviation showed that the method was suitable for its purpose and should be validated by a collaborative trial. I nternational honey standards are specified in a European Honey Directive and in the Revised Codex Standard for Honey (1–3). The standards include criteria for quality factors such as moisture, ash, acidity, hydroxymethylfurfural (HMF) concentration, apparent reducing sugars, and apparent sucrose. HMF is a cyclic aldehyde produced by self-catalytic degradation of sugars, starting mainly from the dehydration of fructose and glucose in an acidic medium (4). It is a major quality factor in honey, and its presence was once considered to be indicative of adulteration with commercial invert syrup. However, it was found that HMF can occur naturally in honey through exposure to heat from improper processing or during extended storage. The amount of HMF produced in honey during storage depends on the pH of the honey and the storage temperature, with greater amounts of HMF produced in honeys from countries with higher ambient temperatures (5). The recent European Union (EU) Directive 2001/110 and the Honey (England) Regulations 2003 (SI 2003/2243) state a maximum HMF level of 40 mg/kg for all honeys except Baker’s honey, which is suitable only for industrial use or as an ingredient in other foods that are then processed and must contain HMF at £80 mg/kg. The Revised Codex Standard also specifies an HMF limit of 40 mg/kg for honey after processing and blending. For honeys from countries with tropical ambient temperatures, and for blends of these honeys, both standards specify a limit of 80 mg/kg. Spectroscopic and chromatographic methods can be used for the determination of HMF in honey, but problems have been reported. The colorimetric method first described by Winkler (6) measures HMF defined as constituents of honey that combine with barbituric acid and p-toluidine under test conditions, and the color of the resulting solution is measured against a blank at 550 nm. This procedure was collaboratively tested in 1979 by AOAC INTERNATIONAL (along with a UV method) and again in 1988 in the United Kingdom (7, 8). The latter collaborative trial incorporated calibration with standard solutions of HMF to calculate the results. Neither trial produced satisfactory results and, in both cases, further work to investigate the method or find an alternative was recommended. In the case of the AOAC trial, comparison of the colorimetric and UV methods revealed that the 2 methods did not give comparable results, and neither was adopted as an official method. The Winkler method has also been criticized for use of the carcinogen p-toluidine, the instability of the developed colored solution, and the temperature dependence of the technique. To overcome these problems, a spectrophotometric method (the White method) was developed that claimed to have retained the accuracy of the chemical method but also to have incorporated the precision of the UV method (9). The method is based on the determination of the UV absorbance of HMF at 284 nm. To avoid interference from other components, the difference between the absorbance of a clear DRIFFIELD ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 1, 2005 121 Received February 12, 2004. Accepted by SG June 3, 2004. Corresponding author's e-mail: m.driffield@csl.gov.uk. Downloaded from https://academic.oup.com/jaoac/article/88/1/121/5657399 by guest on 12 July 2021