Authentication of beeswax (Apis mellifera) by high-temperature gas chromatography and chemometric analysis Miguel Maia a , Fernando M. Nunes b,⇑ a Apismaia, Beekeeping Service, Estrada Municipal 1221, No. 62, 5000-027 Vila Real, Portugal b CQ – Chemistry Research Centre, Chemistry Department, University of Trás-os-Montes e Alto Douro, 5000-801 Vila Real, Portugal article info Article history: Received 7 June 2012 Received in revised form 4 September 2012 Accepted 4 September 2012 Available online 13 September 2012 Keywords: Apis mellifera Authentication Beeswax Paraffin Pattern recognition abstract Chemical characterization and authentication of beeswax of Apis mellifera was performed by high tem- perature capillary gas chromatography coupled to electron impact mass spectrometry or to flame ionisa- tion detection and chemometric analysis. Many major components (>50) of beeswax, odd and even hydrocarbons, oleofin, palmitate, oleate and hydroxypalmitate monoesters were detected, and for the first time palmitate and oleate monoesters esterified with 1-octadecanol and 1-eicosanol are reported to be present in beeswax. Unsupervised pattern recognition procedures, cluster analysis and principal component analysis, were used to find data patterns and successfully differentiate authentic and paraffin adulterated beeswax based on the chemical profile obtained. Independent assessment of beeswax quality and performance of the unsupervised classification methods were performed using classical analytical parameters. The discrimination power of the chemometric unsupervised methods for detection of paraf- fin adulterated beeswax was superior to the discriminating power of classical analytical parameters. Using linear discriminant analysis, classification rules for authentic and paraffin adulterated beeswax samples were developed. The model was validated by leave-one-out cross validation and showed good recognition and prediction abilities, 100% and 99%, respectively. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The quality of beeswax is an important factor in beekeeping in or- der to preserve the natural properties of some beekeeping products and to avoid the rejection of foundation beeswax sheets by the bees. The high commercial value of beeswax makes its adulteration with fatty products, especially with paraffin, an occurrence. It is not sur- prising that several studies have been conducted in order to find authentication parameters for beeswax, including the use of physi- cochemical parameters (Bernal, Jiménez, del Nozal, Toribio, & Martín, 2005; Serra, 1990; Tulloch, 1973, 1980; Tulloch & Hoffman, 1972; White, Riethof, & Kushnir, 1960) and high-temperature gas chromatography with flame ionisation detection (Aichholz & Lorbeer, 1999; Bonvehi & Bermejo, 2012; Jiménez, Bernal, Aumente, Toribio, & Bernal, 2003; Jiménez, Bernal, del Nozal, Martín, & Bernal, 2006; Jiménez, Bernal, del Nozal, Martín, & Toribio, 2009; Jiménez, Bernal, del Nozal, Toribio, & Bernal, 2007; Jiménez et al., 2004; Serra, 1988; Tulloch, 1972, 1980). Analysis of the chemical composition of beeswax presents a huge challenge due to the diversity of compo- nents of lipid nature. Beeswax is mainly composed by a mixture of hydrocarbons, free fatty acids, monoesters, diesters, triesters, hydroxy monoesters, hydroxy polyesters, fatty acid polyesters and some unidentified compounds. Each class of compounds consists of a series of homologues differing in chain length by two carbon atoms. The concentration guide-value approach developed recently (Jiménez et al., 2007) has been shown to have a good performance in the detection of various types of beeswax adulteration and to be able to detect percentages higher than 1–4% of each adulterant (paraffins of different melting points, cow tallow, stearic acid and carnauba wax) (Jiménez et al., 2009). This approach has some significant drawbacks, as there is the need to perform the calculation of the amount of a high number (102) of concentrations of beeswax endog- enous components, using various chemical procedures (Jiménez et al., 2007, 2009). Nevertheless the establishment of the authentic beeswax composition is not a simple task. During the normal com- mercial cycle of beeswax, beekeepers sell the beeswax from their old combs or the capping beeswax, or a mixture of both, to industries for its recycling (Barros, Nunes, & Maia, 2009). Beeswax is then ex- tracted, cleaned and purified by melting (either by boiling the old combs in water, by steam, or by heat from electrical or solar power) followed by sedimentation (Bogdanov, 2004). Purified beeswax is then marketed as foundation beeswax sheets to be placed in empty combs for the beehives. Nevertheless, as beeswax ages and darkens its n-alkane composition changes (Namdara, Neumannc, Sladezkid, Haddade, & Weinera, 2007). The amount of even numbered n-alkanes (C22–C32) increases for darker coloured as compared to 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.09.003 ⇑ Corresponding author. Tel.: +351 259350242. E-mail address: fnunes@utad.pt (F.M. Nunes). Food Chemistry 136 (2013) 961–968 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem