2.1. Chemicals and Reagents. Reference standard heavy metals such as Cd, Cr, Pb, Cu, Fe, Zn, and Mn were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). The chemicals used for digestion analysis were hydrochloric acid (HCl), nitric acid (HNO3), sulfuric acid (H2SO4), and hydrogen peroxide (H2O2); they were of analytical grade and they were purchased from Merck (Darmstadt, Germany).
2.2. Honey Samples. During this study, eighteen honey samples from three different floral types, namely, khalsi (Aegiceras corniculatum), mustard (Brassica juncea), and litchi (Litchi chinensis), were investigated. The samples were collected directly from the comb and local apiary of different regions in Bangladesh between March 2013 and January 2014. At least 100 g of each honey sample was collected under sterile air in a tight glass bottle, labeled properly, and stored at 4- 50 C until analysis. Two honey samples (manuka honey from New Zealand and tualang honey from Malaysia) were used as gold standards to compare their values with those from Bangladesh. The honeys were used as standards due to their well-established chemical and biological properties. Manuka honey is a monofloral honey produced in New Zealand and Australia from the nectar of the manuka tree (Leptospermum scoparium) while tualang honey is a wild multifloral honey produced by the Apis dorsata bees. The bees collect nectar from plants and blossoms from a tall Koompassia excelsa tree, known locally as the “tualang tree,” in which the bees build their hives.
2.3. Physicochemical Parameters 2.3.1. pH. The pH values of the honey samples were determined by using a pH meter (Cole-Parmer, Illinois, USA). A 10% (w/v) honey solution was prepared in fresh Milli-Q water. The pH of each honey was measured on the same day, and the experiments were conducted in duplicate for each sample.
2.3.2. Ash Content. Approximately 1 g of honey sample was transferred into a porcelain crucible and heated for approximately 6 h at 4500 C. Following complete ashing (the ash became white and grayish white), the samples were cooled in a desiccator and weighed. The ash content was calculated by using the following formula:
Ash (%) = Weight of sample after ashing / Weight of fresh sample x 100
2.3.3. Moisture Content. Approximately 3 g of honey was transferred into a porcelain crucible and heated for 3 h in an oven at 1050 C. To ensure the complete removal of moisture, each crucible was reheated and weighed until the weight became constant. The moisture content was calculated by using the following formula:
Moisture (%) = Weight of fresh sample − Weight of dry sample / Weight of fresh sample x 100
2.3.4. Electrical Conductivity. The EC was determined according to the method established by the International Honey Commission. The EC was measured at 200C in a 20% (w/v) solution (dry matter basis) in distilled water by using a Hach conductivity meter. The result was expressed in millisiemens per centimeter (mS⋅cm−1).
2.3.5. Total Sugar Content. The total sugar contents of the honeys were determined by using a refractometer (Delta Refractometer, Code 20–70, Bellingham + Stanley Ltd., England). The sugar content is represented by Brix.
2.3.6. Sample Preparation for Metal Analysis. The collected honey samples were prepared by a High-Temperature Dry Oxidation method [9]. Each honey sample (1 g) was dried in a porcelain crucible at 1000 C to its dry weight, which was then heated to 4450 C for 6 h in an electrical furnace. After complete ashing, 3 mL of HNO3 was added, followed by acid evaporation on a hot plate at 1000 C. Afterwards, 5 mL of HCl was added, and the volume was filled to 10 mL with distilled water (dilution 1: 2). The solution was filtered and preserved in a refrigerator at 4-50 C until further metal analysis with an Atomic Absorption Spectrophotometer, model AA-6300, Shimadzu (Kyoto, Japan), equipped with a Shimadzu model GFA-EX7i graphite furnace atomizer to determine the heavy metals.
The measurement wavelengths for different heavy metals were as follows: Cd (228.67 nm), Cr (357.65 nm), Cu (324.57 nm), Mn (279.43 nm), Pb (217.35 nm), Zn (213.93 nm), and Fe (248.30 nm). Each sample was analyzed in triplicate. Two blanks were injected for each determination. For the calibration curve, standard solutions of each metal solution were prepared at different concentrations (0.0, 0.1, 1.0, 5.0, 10.0, 20.0, and 40.0 g/L). The metal analysis method used for the honey was validated by using a recovery analysis. The percentage recoveries were calculated by using the following equation:
where CE is the experimental concentration that was determined from the calibration curve and CM is the spiked concentration.
2.4. Statistical Analysis. The assays were performed in triplicate, and the results were expressed as the mean values with standard deviations (SD). Correlations were established by using Pearson’s correlation coefficient in bivariate linear correlations (< 0.01). The correlations were calculated with SPSS version 18.0 (IBM Corporation, New York, USA), and the other analyses were performed with Microsoft Excel 2007.