An Approach for Routine Analytical Detection of Beeswax Adulteration Using FTIR-ATR Spectroscopy

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Although beeswax adulteration represents one of the main beeswax quality issues, there are still no internationally standardised analytical methods for routine quality control. The objective of this study was to establish an analytical procedure suitable for routine detection of beeswax adulteration using FTIR-ATR spectroscopy. For the purpose of this study, reference IR spectra of virgin beeswax, paraffin, and their mixtures containing different proportions of paraffin (5 - 95%), were obtained. Mixtures were used for the establishment of calibration curves. To determine the prediction strength of IR spectral data for the share of paraffin in mixtures, the Partial Least Squares Regression method was used. The same procedure was conducted on beeswax-beef tallow mixtures. The model was validated using comb foundation samples of an unknown chemical background which had been collected from the international market (n = 56). Selected physico-chemical parameters were determined for comparison purposes. Results revealed a strong predictive power (R2 = 0.999) of IR spectra for the paraffin and beef tallow share in beeswax. The results also revealed that the majority of the analysed samples (89%) were adulterated with paraffin; only 6 out of 56 (11%) samples were identified as virgin beeswax, 28% of the samples exhibited a higher level of paraffin adulteration (>46% of paraffin), while the majority of the analysed samples (50%) were found to be adulterated with 5 - 20% of paraffin. These results indicate an urgent need for routine beeswax authenticity control. In this study, we demonstrated that the analytical approach defining the standard curves for particular adulteration levels in beeswax, based on chemometric modelling of specific IR spectral region indicative for adulteration, enables reliable determination of the adulterant proportions in beeswax.

Aichholz R., Lorbeer E. (1999) Investigation of combwax of honeybees with high-temperature gas chromatography and high-temperature gas chromatography-chemical ionization mass spectrometry I. High-temperature gas chromatography. Journal of Chromatography A 855(2): 601-615. DOI: 10.1016/S0021-9673(99)00725-6

Aichholz R., Lorbeer E. (2000) Investigation of combwax of honeybees with high-temperature gas chromatography and high-temperature gas chromatography-chemical ionization mass spectrometry. II: High-temperature gas chromatography-chemical ionization mass spectrometry. Journal of Chromatography A 883(1-2): 75-88. DOI: 10.1016/S0021-9673(00)00386-1

Bernal J. L., Jiménez J. J., del Nozal M. J., Toribio L., Martín M. T. (2005) Physico-chemical parameters for the characterization of pure beeswax and detection of adulterations. European Journal of Lipid Science and Technology 107(3): 158-166. DOI: 10.1002/ejlt.200401105

Berry A. J., Delaplane S. K. (2001) Effects of comb age on honey bee colony growth and brood survivorship. Journal of Apicultural Research 40(1): 3-8. DOI: 10.1002/ ejlt.200401105

Beverly M. B., Kay P. T., Voorhees K. J. (1995) Principal component analysis of the pyrolysis mass spectra from African, Africanized hybrid and European beeswax. Journal of Analytical and Applied Pyrolysis 34(2): 251-263. DOI: 10.1016/0165-2370(95)00891-H

Birshtein V. Y, Tul‘chinskii V. M. (1977) Determination of beeswax and some impurities by IR spectroscopy. Chemistry of Natural Compounds 13(2): 232-235.

Bogdanov S. (2004a) Beeswax: quality issues today. Bee World 85(3): 46-50.

Bogdanov S. (2004b) Quality and standards of pollen and beeswax. Apiacta 38: 334-341.

Bogdanov S. (2009) Beeswax: Production, properties, composition and control. Beeswax book. Bee Product Science. 17 pp.

Breed M. D., Garry M. F., Pearce A. N., Hibbard B. E., Bjostad L. B., Page R. E. (1995) The role of wax comb in honeybee nestmate recognition. Animal Behaviour 50(2): 489-496. DOI: 10.1006/anbe.1995.0263

Buchwald R., Breed M. D., Bjostad L., Hibbard B. E., Greenberg A. R. (2009) The role of fatty acids in the mechanical properties of beeswax. Apidologie 40(5): 585-594. DOI: 10.1051/apido/2009035

D’Ettorre P., Wenseleers T., Dawson J., Hutchinson S., Boswell T., Ratnieks F. L. W. (2006) Wax combs mediate nestmate recognition by guard honeybees. Animal Behaviour 71(4): 773-779. DOI: 10.1016/j.anbehav.2005.05.014

DGF-M-V-6 (1957) DGF - Einheitsmethoden - Abteilung M - Wachse. German Standard -Beeswax. 29 pp. Available at:

Edwards H. G. M., Farwel D. W., Daffner L. (1996) Fouriertransform Raman spectroscopic study of natural waxes and resins. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 52(12): 1639-1648. DOI: 10.1016/0584-8539(96)01730-8

Council of Europe (2007) European Pharmacopoeia. 6th Edition. Council of Europe. Strasbourg, Cedex, France. Volume 2. 1085-3308 pp.

Fröhlich B., Riederer M., Tautz J. (2000) Comb-wax discrimination by honeybees tested with the proboscis extension reflex. Journal of Experimental Biology 203(10): 1581-1587.

Jiménez J. J., Bernal J. L., Aumente S., Toribio L., Bernal J. (2003) Quality assurance of commercial beeswax II. Gas chromatography-electron impact ionization mass spectrometry of alcohols and acids. Journal of Chromatography A 1007(1-2): 101-116. DOI: 10.1016/S0021-9673(03)00962-2

Jiménez J. J., Bernal J. L., del Nozal M. J., Martín M. T., Bernal J. (2006) Sample preparation methods for beeswax characterization by gas chromatography with flame ionization detection. Journal of Chromatography A 1129(2): 262-272. DOI: 10.1016/j.chroma.2006.06.098

Jiménez J. J., Bernal J. L., del Nozal M. J., Martín T., Toribio L. (2009) Identification of adulterants added to beeswax: Estimation of detectable minimum percentages. European Journal of Lipid Science and Technology 111(9): 902-911. DOI: 10.1002/ejlt.200800263

Jiménez J. J., Bernal J. L., del Nozal M. J., Toribio L., Bernal J. (2007) Detection of beeswax adulterations using concentration guide-values. European Journal of Lipid Science and Technology 109(7): 682-690. DOI: 10.1002/ ejlt.200600308

Jiménez J. J., Bernal J. L., Aumente S., del Nozal M. J., Martín M. T., Bernal J. (2004) Quality assurance of commercial beeswax. I. Gas chromatography- electron impact ionization mass spectrometry of hydrocarbons and monoesters. Journal of Chromatography A 1024 (1-2): 147-154. DOI: 10.1016/j.chroma.2003.10.063

Maia M., Nunes F. M. (2013) Authentication of beeswax (Apis mellifera) by high-temperature gas chromatography and chemometric analysis. Food Chemistry 136(2): 961-968. DOI: 10.1016/j.foodchem.2012.09.003

Maia M., Barros A. R. N. A., Nunes F. M. (2013) A novel, direct, reagent-free method for the detection of beeswax adulteration by single-reflection attenuated total reflectance mid-infrared spectroscopy. Talanta 107: 74-80. DOI: 10.1016/j.talanta.2012.09.052

MatLab 7.11 (2010b) MathWorks Inc.

Muscat D., Tobin M. J., Guo Q., Adhikari B. (2014) Understanding the distribution of natural wax in starch-wax films using synchrotron-based FTIR (S-FTIR). Carbohydrate Polymers 102: 125-135. DOI: 10.1016/j.carbpol.2013.11.004

Semkiw P., Skubida P. (2013) Comb construction and brood development on beeswax foundation adulterated with paraffin. Journal of Apicultural Science 57(1):75-83. DOI: 10.2478/jas-2013-0009

Serra Bonvehi J. S., Orantes Bermejo F. J. (2012) Detection of adulterated commercial Spanish beeswax. Food Chemistry 132(1): 642-648. DOI: 10.1016/j.foodchem.2011.10.104

Statistica ver. 7. (2004) StatSoft Inc.

Tulloch A. P. (1973) Factors affecting analytical values of beeswax and detection of adulteration. Journal of the American Oil Chemists Society 50(7): 269-272.

University of Tartu (Estonia), Institute of Chemistry. Database of ATR-IR spectra of materials related to paints and coatings. Available at:

Wallner K. (2005) Foundation causing honeybee brood damage. In: Proceedings of ICPBR 9. International Symposium. York - United Kingdom. 12-14 October 2005: 30.

Waś E., Szczęsna T., Rybak-Chmielewska H. (2014) Determination of beeswax hydrocarbons by gas chromatography with a mass detector (GC-MS) technique. Journal of Apicultural Science 58(1): 145-157. DOI: 10.2478/JAS-2014-0015

Winston M. L. (1991) The Biology of the Honey Bee. Harvard University Press. Cambridge. 36 pp.

Zimnicka B., Hacura A. (2006) An Investigation of Molecular Structure and Dynamics of Crude Beeswax by Vibrational Spectroscopy. Polish Journal of Environmental Studies 15(4A): 112-114.

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