Analytical methods

All chemicals were analytical grade or better. Ultrapure water was used to prepare of solutions and dilutions produced by a Milli-Q water purification system (Millipore S.A.S., Molsheim, France).

The digestion of samples for element analysis was carried out according to the method of Kovács et al. (1996), which has been validated using animal and plant materials in our accredited laboratory. A total of 3 g honey was added to 10 ml nitric acid (69% v/v; VWR International Ltd., Radnor, USA) and the samples allowed to stand overnight. Samples were pre-digested at 60 °C for 30 min. After cooling, 3 ml hydrogen-peroxide (30% v/v; VWR International Ltd., Radnor, USA) was added and the samples heated at 120 °C for 90 min. After digestion, ultrapure water was added to make a final volume of 50 ml. Samples were homogenized and filtered using qualitative filter papers (Sartorius Stedim Biotech S.A., Gottingen, Germany). The concentrations of boron, potassium, magnesium, sodium, phosphorus and sulphur were determined by ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) (Thermo Scientific iCAP 6300, Cambridge, UK). The applied wavelengths were the following: 249.772 for B, 769.896 for K, 279.806 for Mg, 818.326 for Na, 213.617 for P and 182.563 for sulphur. Detection limits (DL) of ICP-OES were: 0.0004 mg/kg for B, 0.527 mg/kg for K, 0.104 mg/kg for Mg, 0.009 mg/kg for Na, 0.489 mg/kg for P and 0.108 mg/kg for S. The determination of arsenic, cadmium, chromium, iron and zinc was carried out using ICP-MS (Inductively Coupled Plasma Mass Spectrometry) (Thermo Scientific XSeries 2, Bremen, Germany). Measured isotopes (amu) were as follows: 75 for As, 111 for Cd, 52 for Cr, 56 for Fe and 66 for Zn. Detection limits (DL) of ICP-MS were: 0.019 µg/kg for As, 0.003 µg/kg for Cd, 0.38 µg/kg for Cr, 0.017 µg/kg for Fe and 0.004 µg/kg for Zn.

Moisture contents (in %) were determined by refractometry (AOAC 1995a, 969.38) using a Medline DIGIT 5890 ATC Honey Pocket refractometer (United Kingdom). Electrical conductivity (EC, in µS/cm) was determined according to the method of Bogdanov et al. (1997) in a 20% (w/v) solution of honey (in distilled water) using a conductometer (FiveEasy™ FE30, Mettler-Toledo AG, Switzerland). The pH values were measured in a 30% (w/v) solution of honey (in distilled water) with a pH meter (FiveEasy™ FE20, Mettler-Toledo AG, Switzerland), according to the MSZ 6943-3:1980 standard.

Diastase activity was determined following the spectroscopic method of Bogdanov (2009), using a spectrophotometer (Evolution 300 LC, Thermo Electron Corporation, England) at 660 nm. Applied reagents were iodine (VWR International BVBA, Leuven, Belgium), potassium iodide, sodium acetate, acetic-acid, sodium chloride and starch (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Diastase activity is expressed as a diastase number (DN). Determination of the hydroxyl-methyl-furfural (HMF) content of samples in mg/kg was based on the White method (Bogdanov 2009). Applied reagents were potassium hexacyanoferrate (II) trihydrate for Carrez I. and zinc acetate for Carrez II (VWR International BVBA, Leuven, Belgium) and sodium disulfite (AppliChem GmbH, Darmstadt, Germany). Proline content in mg/kg was measured using a spectrometric assay (Bogdanov 2009) with a spectrophotometer (Evolution 300 LC, Thermo Electron Corporation, England) at 510 nm. Applied reagents were formic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), ninhidrin, methoxyethanol, l-proline (Alfa Aesar GmbH & Co. KG, Karlsruhe, Germany) and 2-propanol (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). TPC in mg gallic acid equivalent (GAE)/100 g was determined according to the Folin-Ciocalteu method (Meda et al. 2005). The absorbance of blue-colour complex was measured at 760 nm. Applied reagents were 3,4,5-trihydroxybenzoic acid (Alfa Aesar GmbH & Co. KG, Karlsruhe, Germany), sodium carbonate and methanol (Scharlab S.L., Spain), Folin-Ciocalteu reagent (VWR International S.A.S., France).

The colour of honey samples was determined by spectrophotometric measurement from a 50% (w/v) honey solution at 635 nm (White, 1984). The honeys were classified according to the Pfund scale after conversion of the absorbance values:

Sucrose, fructose and glucose contents were determined based on AOAC (1995b) 977.20 methods with HPLC. Applied chemicals were acetonitrile as a mobile phase (VWR International S.A.S., France), sucrose, fructose and glucose standard solution (Alfa Aesar, Thermo Fisher GmbH, Germany). Instruments and equipment used were a chromatograph (Merck Hitachi L6200A, Germany), detector (Merck LaChrom RI Detector L-7490, Germany), column (Phenomenex Luna 5µ NH2 100A, USA), sample clarification kit (PALL A/B Glass 13 m, Sigma-Aldrich Kft., Budapest, Hungary) and syringes (Hamilton MIcroliter^® # 710, Switzerland).