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Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey

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Abstract

5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg−1 and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg−1. Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg−1 (15 %). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n = 6), mean intra-day precision 3.2 % (n = 3 within a plate; n = 2 repeated within a day), mean inter-day precision 3.7 % (n = 3), mean reproducibility over the whole procedure including sample preparation 4.1 % (n = 2), and mean recovery 106.9 % (n = 5 different concentrations; n = 4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2 %. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8 %. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.

HPTLC separation of HMF from honey matrix for honey with very low HMF content

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Acknowledgments

Professor Lubomír Opletal, ADINACO Research Group, Charles University, Prague, Czech Republic, is thanked for granting leave to AH. The project was partially co-financed by the European Social Fund and the state budget of the Czech Republic, TEAB, project no. CZ.1.07/2.3.00/20.0235 and SVV UK 267 002. Merck Millipore, Darmstadt, Germany, is thanked for support with the Reflectoquant assay and HPTLC plates.

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Authors and Affiliations

  1. ADINACO Research Group, Department of Pharmaceutical Botany and Ecology, Charles University, Heyrovskeho 1203, 500 05, Hradec Kralove, Czech Republic

    Anna Hošťálková

  2. Institute of Nutritional Science, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392, Giessen, Germany

    Ines Klingelhöfer & Gertrud E. Morlock

Authors
  1. Anna Hošťálková
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  2. Ines Klingelhöfer
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  3. Gertrud E. Morlock
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Correspondence to Gertrud E. Morlock.

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Anna Hošťálková and Ines Klingelhöfer contributed equally to this work, which was performed at JLU Giessen.

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Hošťálková, A., Klingelhöfer, I. & Morlock, G.E. Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey. Anal Bioanal Chem 405, 9207–9218 (2013). https://doi.org/10.1007/s00216-013-7339-6

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  • DOI: https://doi.org/10.1007/s00216-013-7339-6

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