2.1. Honey Samples. Twelve honey samples were collected from several different geographical areas of Bangladesh between January 2012 and March 2014. Eight of the samples were raw honeys, while four were commercial honeys. The raw samples were collected directly from the comb as well as the local apiary by experienced honey collectors while the commercial honey samples were purchased from a departmental store in Dhaka, Bangladesh. Among the eight raw honey samples, five were multifloral and three were monofloral as confirmed by the suppliers. As for the commercial honey, three were multifloral while one was monofloral. All samples were stored in glass flasks at low temperature (40 C) before analysis. All samples were analyzed within six months of collection.
2.2. Chemicals and Reagents. Gallic acid, benzoic acid, catechin, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,4,6-tris(2- pyridyl)-1,3,5-triazine (TPTZ) were purchased from SigmaAldrich (St. Louis, Missouri, USA). All standards were purchased from Sigma-Aldrich Production, GmBH, Switzerland. All of the chemicals and reagents used in this study were of analytical grade.
2.3. Sample Digestion for the Mineral and Trace Element Content. The major and trace elements in the honey samples were analyzed following the standard method [19] with slight modifications. Briefly, 2 g of each honey sample was digested with perchloric acid (HClO4) and hydrochloric acid (HCl) in a ratio of 1: 2 (V/V) on a hotplate (LMS1003, Daihan Labtech Corporation Ltd., Korea) to near dryness with the temperature controlled at 4000 C. The sample containing volumetric flask was cooled at room temperature before the addition of deionized water to the mark on the flask.
For the measurement of sodium, potassium, and magnesium, 100-fold diluted samples were filtered using a nylon membrane filter (Whatman, 0.8 ?m pore size, 47 mm diameter) and preserved for the determination of mineral and trace element content.
2.4. Mineral Analysis. In the present study, four major cationic elements, such as sodium (Na), potassium (K), calcium (Ca), and magnesium (Mg), and seven trace elements, including iron (Fe), zinc (Zn), copper (Cu), cadmium (Cd), molybdenum (Mo), cobalt (Co), and nickel (Ni), and three heavy metals, such as lead (Pb), mercury (Hg), and arsenic (As), were determined in the honey samples using an atomic absorption spectrophotometer (AA-7000, Shimadzu Corporation, Kyoto, Japan) coupled with an autosampler ASC 7000. Specifically, the element As was measured using the graphite furnace technique, and Hg was measured using cold vapor atomic absorption spectrometry; all of the other elements were measured using a direct absorption technique according to the standard guidelines of the manufacturer.
2.5. Instrumentation. A blank solution was prepared under similar conditions, and the mean signal of the blank solution was subtracted from the analytical signals of the honey samples, as automatically performed by the instrument. Background correction (BCG) was performed using the BGC-D2 (D2 lamp method; high speed, dual-frequency simultaneous measurement) method. The optimum analytical range was 0.5 to 1.0 absorbance units, with coefficients of variation that ranged between 0.05 and 0.40%. Determination was made on a dry weight basis for all of the samples. The results were calculated and expressed as mg/kg of honey.
2.6. Preparation of Standards and Calibration Curve. The standard stock solutions of Na, K, Mg, Fe, Ca, Zn, Cu, Pb, Cd, As, Mo, Co, Ni, and Hg were prepared (1000 mg/L), and the number of minerals in each sample type was calculated based on the slope of the standard curve, which was prepared as previously described.
2.7. Quality Control and Quality Assurance. The method was validated for accuracy and precision in accordance with the European Commission (EC) guidelines. The accuracy was measured by analyzing and comparing the samples with measured values of known spiked samples. The precision was expressed as the relative standard deviation (RSD). The percentage recovery was calculated using the following formula: Percentage recovery = [CE/CM × 100], where CE is the experimental concentration determined from the calibration curve and CM is the spiked concentration. The mean percentage recovery of the method ranged from 88.0% to 105.0%, while the mean precision ranged from 4% to 10%.
2.8. Antioxidant Properties. The total polyphenol (TPP) content of the honey samples was estimated by spectrometric determination based on Folin-Ciocalteu’s method using a PD-303S spectrophotometer (APEL, Japan). The TPP content was determined in terms of gallic acid equivalents (GAEs) and expressed as mg of GAEs/kg of honey. The total flavonoid (TF) content was estimated using an aluminum chloride colorimetric assay. The concentration of TF was determined by catechin equivalents (CEs) and the results were expressed as mg of CEs/kg of honey. The ascorbic acid (AA) content of the honey samples was estimated by a method that was previously established by Omaye et al. The AA content was expressed as mg of ascorbate equivalents (AEs) per g of honey.The reducing sugars content was estimated according to the Nelson-Somogyi method. The reducing sugar content was expressed as g of D-glucose per 100 g of honey.