3.2.1. Method Validation

The method was validated according to Eurachem criteria [41]. Commercial standards were used for the evaluation of method linearity, limits of detection (LODs), limits of quantification (LOQs), accuracy, repeatability, and intermediate precision. LODs and LOQs were calculated as 3.3 σ/S and 10 σ/S, respectively, where σ is the standard deviation of six blanks and S is the slope of the relative calibration curve. A good linearity was obtained for all elements investigated with R2 values ranging from 0.9992 (for Cu and Se) to 0.9999 (for V). The limits of detection (LODs) ranged from 0.001 to 0.051 μg kg−1, and the limits of quantification (LOQs) ranged from 0.003 to 0.168 μg kg−1. The lowest average recovery was observed for mercury with 92.93%, while the highest was obtained for strontium with 103.03%. Accuracy was assessed by evaluating six determinations on certified reference materials (NIST1570A spinach leaves) and was reported as the percent recovery between the value found with the calibration curve and the true value reported in the certified reference materials. If the element was not certified in the reference materials, the matrix was spiked with the known amount of analyte, and was analyzed following the procedures discussed previously. Based on these results, the analytical characteristic (linearity, sensitivity, and accuracy) can be considered to be satisfactory for the purposes of the analysis (Table 3).


**Table 3.** Analytical parameters for method validation.

\* Not present in the certified matrix. Element added for method validation.

## 3.2.2. Mineral Contents

The content of inorganic elements in the samples, labelled with letters from A to O, are shown in Table 4 and reported as average values and standard deviations.


**Table 4.** Trace elements (**a**), potentially non-toxic elements (**b**), and potentially toxic elements (**c**) present in dried spices. The contents are expressed as the mean value (mg kg −1) and standard deviation.

A, black cumin seeds; B, Iranian Tokhme Sharbati; C, clove buds; D, Shahjeera; E, Abbaszadeh safron; F, organic fenugreek; G, whole black pepper; H, cinnamon; I, Abthul Ahmar (Asario); L, Ajwan seeds; M, whole coriander seeds; N, black sesame seeds; O, Sabja seeds.

Although various classifications for trace elements have been proposed and may be controversial, this paper uses the World Health Organization recommendations which classifies trace elements as: essential trace elements, potentially non-toxic essential elements, and potentially toxic elements [42].

## 3.2.3. Trace Elements

Given the increasing consumption of spices and aromatic herbs in the daily diet, it is also interesting to elucidate the content of minerals known for their nutritional roles such as Cr, Fe, Mn, Cu, Se, and Zn. Several studies on the content of mineral and trace elements in spices and herbs have remarked that they occur in a wide range of concentrations [43–46].

Data on Cr content in these products are needed. Chromium has been quantified in several foods and beverages, and currently the most comprehensive source are the Danish food composition tables [47]. The content of chromium in foods is relatively low and most foods present a content below 0.1 mg kg−1. Data from the literature indicate that the presence of Cr in spices and aromatic herbs is higher than other foods and beverages, within a range from 0.01 to 3.0 mg kg−<sup>1</sup> [48–51]. In our study, the chromium level found in spices was below 0.3 mg kg−1, except for the high content found in cinnamon (2.35 mg kg−1) and thymol seeds (1.11 mg kg−1).

Selenium content varied from 0.003 to 0.505 mg kg−1; in particular, selenium was reported with values: 0.254, 0.287, 0.254, 0.505, and 0.485 mg kg−<sup>1</sup> for caraways, cress sprouting seeds, thymol seeds, coriander, and black sesame seeds, respectively. Selenium (Se) is an essential trace element involved in the synthesis of various selenium-containing proteins, and also has other relevant biological functions; moreover, it has a fundamental role in the human diet since it may act as a preventive agen<sup>t</sup> against some health conditions [51,52].

#### 3.2.4. Potentially Non-Toxic Elements

Eight trace elements that are not normally known for their toxic effects were identified, but it is nonetheless important to monitor them, because if very high concentrations of these metals are ingested, they can lead to physiological disorders [53,54]. Their maximum concentrations were found to be in the decreasing order as follows: Sr > Ni > Sn > V > Co > Sb > Be. All these potentially non-toxic trace elements were contained in variable amounts in the analyzed spices. In particular among these, the major trace elements were: Sr, which ranged from 95.92 ± 0.20 mg kg−<sup>1</sup> (carraway) to 4.30 ± 0.02 mg kg−<sup>1</sup> (fenugreek); Ni, which ranged from 3.05 ± 0.12 mg kg−<sup>1</sup> (black cumin) to 0.42 ± 0.01 mg kg−<sup>1</sup> (chia seeds); Sn, which ranged from 2.39 ± 0.04 mg kg−<sup>1</sup> (coriander) to 0.11 ± 0.04 mg kg−<sup>1</sup> (clove buds); and Co, which ranged from 0.29 ± 0.00 mg kg−<sup>1</sup> (thymol seeds) to 0.02 ± 0.00 mg kg−<sup>1</sup> (black pepper).

#### 3.2.5. Potentially Toxic Elements

Concerning Cd, Pb, As, and Hg, the following ranges have been observed in the 13 analyzed spices: Cd (0.003–0.079 mg kg−1), Pb (0.008–0.544 mg kg−1), As (0.003– 0.339 mg kg−1), and Hg (0.001–0.010 mg kg−1). The contamination level of the analyzed samples followed the sequence: Pb > As > Cd > Hg.

Heavy metals should be closely monitored, considering that the absorption and bioaccumulation of those compounds, with reference to their toxic and mutagenic effects, have a negative effect on consumers' health [8,44,45,55].

According to the European Commission Regulation (EC) No. 1881/2006 and its amendments, Regulation (EU) No. 1317/2021 and Regulation (EU) No. 1323/2021, the maximum levels in fresh herbs for lead and cadmium have been set at 0.1 mg kg−<sup>1</sup> and 0.2 mg kg−1, respectively. However, for many elements, there is a lack of shared worldwide regulation, and reference could be made to the values reported by WHO and EFSA, namely 5.0 and 0.2 mg kg−<sup>1</sup> for As and Hg, respectively [28–36,56,57].

Concerning the content of potentially toxic minerals (Cd, Pb, As, and Hg), the results showed that all the spices analyzed did not present contamination concerns for cadmium, arsenic, and mercury, as the contents of these three metals were always lower than the permitted limit values established by the European Commission regulation and WHO. On the other hand, for Pb content, two samples showed two warning values with respect to the established permitted limit of 0.1 mg kg−1. Ajwan seeds from India (sample L) showed

a lead content (0.544 mg kg−1) five times higher than the permitted limit (*p* < 0.05) while the lead content found in Abbaszadeh saffron from Iran (0.096 mg kg−1, sample E) was close to the accepted limit (*p* > 0.05), indicating that for these samples, there was a possible threat to health.

Several studies have reported a potential threat to the nervous system from aluminum [58,59]. The content of aluminum ranges from 7.112 mg kg−<sup>1</sup> in fenugreek to 46.889 mg kg−<sup>1</sup> in chia seeds, with the exception of carraway, saffron, and thymol seeds, in which the reported values were 87.507 mg kg−1, 145.216 mg kg−1, and 930.198 mg kg−1, respectively, showing a high capacity to accumulate aluminum.

Lopez et al. [58] showed data on the levels of Al in 72 dried samples of 17 different spices and aromatic herbs, and aluminum levels ranged from 3.74 to 56.50 mg kg−1. Bratakos et al. found Al values in spices with a mean value of 157 mg kg−<sup>1</sup> [58]. For foods from plant origins, high aluminum concentration could be related to its high content in the soil where the plants are grown, or to the fact that plants grow in acid soils, because its availability depends on soil pH [59,60].
