**5. Analytical methods**

showed good performance in separation and preconcentration of those trace metals with acceptable recovery values (higher than 95%) in comparison with other reported methods [45]. Another methodology with a purpose similar to solid phase extraction is wet digestion, which applies strong acids to digest organic material in honey. Specifically, samples must be heated for 3–4 h at 105°C to remove as much water as possible. Following this, digestion takes place at

organic matter is fully destroyed. Excess acid is then evaporated through drying. Finally, the

measured via AAS, ICP-OES, or ICP-MS [46]. On variation of wet digestion is calcination in a muffle furnace, which produces ashes that can then be suspended in a solution of 0.1 M HNO<sup>3</sup>

ment of diverse analytes through only one approach. However, a disadvantage is the risk for cross-contamination between samples and the time of analysis, so close supervision is needed during the execution of experimental procedures. Another variation on wet digestion that has been implemented with notable success is that of using microwaves to induce wet digestion [49]. Tuzen et al. [50] evaluated the efficiency of calcination with a muffle furnace as compared to other ash-generating techniques, such as wet digestion using inorganic acids and through microwave. For this, various honey samples were assessed and submitted to three digestion procedures. The obtained results for copper, magnesium, zinc, iron, lead, cadmium, and nickel, among others, were classified according to the standard deviation obtained for each measurement. From the resulting values, the authors concluded that microwave digestion gave the best results, followed by direct wet digestion. Finally, calcination via a muffle fur-

Currently, no technique has been validated for determining and measuring metals specifically in honey. The AOAC [37] lists calcination in a muffle furnace as the official method for determining metals in any organic sample. However, the application of this technique to honey is limited due to the chemical properties of distinct metals and the different ranges in which each type of metal can exist in a honey sample. The behavior of any sample during calcination is fundamentally determined by the organic composition of the sample. Preventing losses in the interior of the muffle furnace is a complicated process to control, directly affecting the distribution of the data obtained from muffle furnace measurements. Furthermore, although metals are often collectively referred to as a single group of elements, metals present important physico-chemical differences. These variations constitute another challenge during calcination via a muffle furnace. Specifically, the chances of cross-contamination within the muffle furnace are high, ultimately influencing the distribution of the obtained values.

Likewise, the toxicity to human health presented by metals varies from one element to the next. Some heavy metals, such as lead, mercury, and cadmium, are highly toxic and are found at much lower concentrations than other elements. Although there are not maximum residue levels for these elements, the World Health Organization and Food and Agriculture Organization have established acceptable levels for honey (i.e., Pb: 25 μg/kg ; Hg: 5 μg/kg; and Cd: 7 μg/kg; [51]). Therefore, sample loss during the process of obtaining ash can result in imperceptible differences between the actual and recorded values for the aforementioned

at 3–30% v/v [47, 48]. A noted advantage of this method is that it permits measure-

/HCl 1:1) until the

10% v/v. The resulting solutions can be directly

45°C through the addition of an aliquot composed of an acid mix (i.e., HNO3

obtained ashes are suspended in 10 mL HNO3

nace resulted in the least precise and most disperse results.

and H2 O2

316 Honey Analysis

In recent years, much investigation has been focused on developing new methods for measuring metal concentrations in honey, with the aims of obtaining more reliable and exact values. Electrochemical techniques are one such option and have already shown more sensitive detection limits for some elements. One of these techniques is that samples are subjected to combined acid mineralization and microwave calcination before posterior analyses, with results evidencing good reproducibility for the quantification of copper, lead, cadmium, and zinc concentrations in eucalyptus honey [52]. Similarly, Buldini et al. [53] measured metal concentrations in various types of honey using hydrogen peroxide-mediated digestion and posterior analyte quantification using ionic chromatography or voltamperometry. The results from this method were satisfactory when compared against values obtained for the same samples by traditional methods. Nevertheless, the proposed method was determined only reliable for investigative ends as the large volumes of hydrogen peroxide needed to process each sample translate into a notable risk that would be difficult to implement and manage on an industrial scale. Moreover, higher sample quantities would also be required.

A distinct strategy for the analysis of metals through electrochemical techniques was proposed by Muñoz and Palmero [54]. Specifically, honey samples were diluted in hydrochloric acid, a solution to which gallium nitrate was then added to decrease any interferences that could complicate adequate zinc measurements. This method provided better results not only for zinc, but also for cadmium and lead in the assessed honey. The primary advantage of the technique proposed by Muñoz and Palmero [54] is that digestion through H2 O2 was not used. Nevertheless, this technique was unable to measure elements such as copper, thus limiting its widespread application.

In general, metals are quantified through traditional methods such as AAS, ICP-OES, and ICP-MS due to high instrument sensitivities. While one-third of all honey mineral contents is potassium (K), elements frequently found in trace amounts include iron, copper, and manganese, among others [55, 56]. For the more predominant inorganic elements in honey, AAS is the most convenient measurement method [43, 57]. However, when the elements under study exist in lower concentrations, then the use of more highly sensitive techniques should be preferred, using ICP-MS as the primary option and ICP-OES as the secondary option [51, 58, 59]. In many cases, ICP-MS has been used for determining metals in other related products obtained from bees. The analysis of metal contents in honeybee venom showed that this equipment permits achieving very low levels for quantifying of As, Ba, Pb Cd, Sb and Cu. This tool is important when honeybee venom is a recommended treatment for certain diseases in medicine [60]. Whatever the analysis of honey, pollen, or any other product taken from beehives, it is important to note that the ICP-MS requires several steps to be considered before chemical analysis. In order to achieve reliable results, it is advisable the optimization of the instrument including calibration with standard solutions, fortification of samples, and the use of a reference material. Also, for having a correct validating process for one analytical method, it is necessary to incorporate a confirmation method to obtain quality data. These last analyses may be performed using graphite furnace atomic absorption spectrometry [61].
