**4. Sample treatments**

Most soils are heterogeneous and constituted by different minerals, solids, and organic compounds. Various interaction mechanisms of soil with heavy metals have been described, including diffusion through micropores and adsorption at sites with variable reactivity. It is not possible to discriminate between these mechanisms, being more appropriate to use the term "sorption" in order to describe the retention of heavy metals by these three pathways [24]. The type of sorption and metal-binding mechanisms depend on various factors, such as ionic radius, electronegativity, surface type, valence electrons, and ionic strength of the solution. Currently, strict regulations exist for metals due to residual accumulations and persistence in the environment, as supported by findings after specific contamination events [25, 26]. Furthermore, a number of studies have established the threat posed by the possible contamination of water and soil resources destined for agricultural ends. Any

subsequently produced plants would represent healthy risk to consumers [27–29].

The presence of metals in honey has been associated with the presence of hives close to contamination sources, such as factories, highways, volcanoes, or mines/mine tailings. Contamination sources can also include agrochemicals that contain cadmium and arsenic, among others [30–32]. Due to this association, extensive research has been conducted in honey to determine the relationship between heavy metal contents and quality indicators or biological markers [33, 34]. Frequently, heavy metal concentrations in honey are low, complicating the analysis of these elements. This complication is directly evidenced in the quality of obtained results, where any loss during the analytical processing of samples influences the

Related to the analysis of honey, Przybyłowski and Wilczyńska [36] conducted research on polyfloral honey produced in Poland to evaluate possible relationships between parameters such as pH, the glucose:fructose ratio, moisture, electric conductivity, and hydroxymethylfurfural concentration, among others, and the presence of cadmium, lead, and zinc. These relationships were determined based on methodologies established by the Association of Analytical Communities [37] for processing organic samples and performing posterior metal assessments. While no clear relationships were found between the measured parameters and the metals studied, discrete cadmium and lead concentrations were found in all of the studied samples. This finding indicates a degree of environmental contamination. Similarly, relationships did exist between plant origin and the presence of zinc in samples. Further research was conducted by Hernández et al. [38], who analyzed the metal contents in 81 honey samples from the Canary Islands and compared results against 35 additional samples from zones in Spain and Europe in general. Analyses established that the concentrations of alkaline and alkaline earth metals were within specific ranges that discriminated between Canary Islands and European mainland honey. The authors therefore concluded that this type of analysis can be used to certify the source of a honey. Hernández et al. [38] also suggested that the presence of metals could indicate the production of honey in areas

**3. Metals in honey**

314 Honey Analysis

contaminated by these metals.

concentration values determined for each metal [35].

Before assessing the metal contents in honey, samples need to be pretreated to eliminate the majority of organic matrix components that can interfere in obtaining results. One method used in determining metal contents is solid phase extraction. This method can remove the predominant sugars from honey, thereby allowing for the collection of concentrated metal extracts that can then be analyzed through atomic absorption spectroscopy (AAS), inductively coupled plasma optical emission spectrometry (ICP-OES), or inductively coupled plasma mass spectrometry (ICP-MS).

Solid phase extraction can be useful in fractioning extracts of an element, zinc for example, that could be present in honey as hydrophobic complexes or as cationic species. Resins, such as Amberlite XAD-16 and Dowex-x8-200, must be used in these cases to accurately separate metal species [42]. Other strong cation-exchange styrene-divinylbenzene resins, including Amberlite IRP-69, Dowex 50W x8-400, and Dowex HCR-W2, have been used to determine and fractionate manganese and zinc contents in extracts [43]. Similarly, Dowex 50W x8-400 and Dowex HCR-W2, together with the Diaion WT01S resin, have been used to satisfactorily detect copper and zinc species [44]. Solid phase extraction is advantageous because it destroys all of the organic materials present in honey samples, thereby reducing analysis time and risks of analyte loss that could affect result reliability. However, application of this method is limited when a mix of various metals is needed for subsequent analyses.

Recently, a new chelating resin of poly[2-(4-methoxyphenylamino)-2-oxoethyl methacrylateco-divinylbenzene-co-2-acrylamido-2-methyl-1-propanesulfonic acid] was synthesized for determining Cd(II), Co(II), Cr(III), Cu(II), Fe(III), Mn(II), Pb(II), and Zn(II) ions. This resin 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 45°C through the addition of an aliquot composed of an acid mix (i.e., HNO3 /HCl 1:1) until the organic matter is fully destroyed. Excess acid is then evaporated through drying. Finally, the obtained ashes are suspended in 10 mL HNO3 10% v/v. The resulting solutions can be directly 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> and H2 O2 at 3–30% v/v [47, 48]. A noted advantage of this method is that it permits measurement 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 furnace resulted in the least precise and most disperse results.

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 elements. This is a relevant issue when considering the low maximum residue levels permitted, where any loss can cause statistically significant differences between classifying a honey as contaminated or uncontaminated by these elements.
