Abstract

A closed-vessel microwave digestion method was developed for the simultaneous determination of macro, micro, and toxic elements (Mg, K, Ca, Al, Mn, Fe, Cu, Zn, Se, Rb, V, Cd, and As) in wheat by inductively coupled plasma mass spectrometry (ICP-MS). A two-level factorial design with four variables was employed to determine the optimal conditions for the significant parameters. The optimal conditions for microwave digestion of ground wheat samples were found to be 0.5 g of the sample, 9 mL of 8 mol/L HNO3, and 3 mL of 33% H2O2 at 210°C. The analytical method was validated, and the accuracy of the method was then evaluated through analysis of wheat flour-certified standard reference materials (NIST SRM 1567b). The validated procedure was subsequently applied to the analysis of 12 wheat samples sourced from local farms in the Al-Qassim region and a further six samples purchased from the Al-Qassim markets, covering six imported brands common in Saudi Arabia. The results of the study showed that the level of minerals in wheat grown in desert areas is higher than in other areas. The concentrations of macroelements and microelements in the Al-Qassim samples exceeded those of the imported samples by 12.41% and those of the samples from other countries by 33.90%. The results were also evaluated using multivariate analysis techniques: principal component analysis (PCA) and hierarchical cluster analysis (HCA).

1. Introduction

Flour has an important role in the human diet and is consumed daily in most cultural groups as a key ingredient in various types of food products such as breads, cereals, cakes, cookies, and baked products. Most of the world’s population consumes large volumes of wheat (Triticum aestivum L.) [1, 2], and it is the largest food commodity crop worldwide by volume, with a global production of 626 million tons in 2007. The average worldwide per capita consumption of wheat per annum in 2005 was 68 kg, with 61 kg and 95 kg being consumed in developing and developed countries, respectively [3]. The main wheat producers are China, the European Union, India, and the USA. Thus, wheat cultivation is important in many cultures, both economically and nutritionally [4, 5]. In Saudi Arabia, the popularity of wheat as a food product stems from its versatility, as it is used in pizza dough, different kinds of bread, including hamburger buns, and traditional bakery products. It has been estimated that wheat consumption in Saudi Arabia will remain at 3.5 million tons in 2020-21, taking into account that fewer tourists will be visiting the country because of COVID-19. In the 2020-21 crop year, the Saudi Grains Organization (SAGO) increased the maximum local wheat production cap to 1.5 Mt per year until 2022-23 [6].

Many properties of wheat flour, including flavor, texture, and color, are related to its mineral concentration [7]. Considering its high level of consumption, precise determination of the major, minor, and trace elements in wheat flour is important from both a nutritional and a toxicological perspective [8]. Minerals constitute 1–3% of the weight of a cereal grain and are more concentrated in the external areas of the wheat grain [9]. The trace metal content is highly variable and will depend on the variety, type of land where it has been cultivated, fertilization that has been used, and weather [10]. Numerous research articles reported in the United States Department of Agriculture (USDA) Nutrient Database show that wheat flour is rich in minerals such as calcium, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc, copper, and selenium [7]. Elements are present in various forms in nature, and many are considered essential nutrients, needed for the body to perform cell functions at biological, chemical, and molecular levels. Trace elements mediate vital biochemical reactions by acting as cofactors for many enzymes as well as centers for stabilizing the structures of enzymes and proteins. Some of the trace elements control important biological processes by binding to molecules on the receptor site of cell membranes or by alternating the structure of membranes to prevent the entry of specific molecules into the cell. These trace elements have clinical significance and a dual role. At normal levels, they are important for the stabilization of cellular structures, but in deficiency states, they may stimulate alternate pathways and cause diseases. Consumption of food contaminated with heavy metals may lead to adverse health effects, such as neurological disorders and cancer, and cause damage to important body organs, such as the kidneys, lungs, and liver [11]. Heavy metals such as Pb, Cd, Zn, Cr, and Ni that exist in high concentrations in the soil environment may affect both the nutrient content and enzyme activity in wheat [3]. Exposure to heavy metals should always be below the guidelines recommended by the World Health Organization (WHO) [12].

Trace element concentrations can be determined using various analytical methods [13]. One of the established techniques used in the determination of minerals is inductively coupled plasma mass spectrometry (ICP-MS), which allows simultaneous multielement analysis with good sensitivity and a wide quantification range [14]. Conventional multielement determination by ICP-MS techniques requires the sample to be converted to a solution. This can be achieved with the use of oxidants and energy sources capable of breaking molecular bonds and crystalline structures, promoting the partial or complete digestion of the solid [15]. The use of dilute acids in microwave-assisted digestion has been shown to be a good alternative method for sample preparation, harnessing the well-known efficiency of diluted reagents for the digestion of organic materials [1517]. When compared with the use of concentrated acids, such an approach improves the purity of blank solutions, thus decreasing the limit of quantification, reducing the amount of waste generated, and reducing the risk of damage to the equipment [16].

Experimental design methods that allow the simultaneous study of several controlled variables are faster to implement and more cost-effective than traditional one-factor-at-a-time (OFAT) univariate approaches. Several experimental design models that reduce the number of experiments can be used in different situations. The experimental variables are explored to determine whether they significantly influence the system under study and find the optimum conditions for the best results [18, 19]. Experimental designs for first-order models (e.g., factorial designs) can be used when the dataset does not present curvature [20].

Multivariate data analysis, such as principal component analysis (PCA) and hierarchical cluster analysis (HCA), is used to process a large amount of data, assisting in the interpretation of the results [21]. In food analysis, these techniques are often used in classification, determination of the geographic origin, and evaluation of the viability of samples according to their chemical properties [2127].

Our objective was to develop, optimize, and validate an analytical method comprising microwave-assisted digestion procedures using a mixture of hydrogen peroxide and dilute nitric acid, followed by ICP-MS, and apply it to determine the concentration of 13 elements in wheat flour samples collected from several regions in Al-Qassim, Saudi Arabia. The selection of appropriate conditions for digestion was based on the use of experimental design methodology. We then compared the mineral and trace element composition of locally grown wheat to that reported in wheat grown in other countries.

2. Materials and Methods

2.1. Instrumentation

An XS105 analytical balance model (Mettler Toledo, Columbus, USA) with a resolution and maximum capacity of 0.00001 g and 120 g, respectively, was used to weigh wheat flour samples prior to digestion. A START D digestion microwave system from Milestone (Germany), equipped with reaction sensors for pressure and temperature control and ten 100 mL PTFE high-pressure digestion vessels, was used for the total digestion of samples. The equipment had power settings up to 1200 W, controlled at up to 300°C in the reference vessel, provided noncontact temperature monitoring and infrared control up to 300°C in all containers, and allowed direct monitoring of pressure up to 100 bar in the reference vessel. Purified water (0.055 μs/cm) was obtained using a Barnstead water purification system ASTM Type II (Thermo Electron LED GmbH, Germany). The mineral elements were determined using inductively coupled plasma mass spectrometry (ICP-MS 7800, Agilent) with the operational parameters listed in Table 1.

2.2. Samples and Reagents

Eighteen samples of wheat grain traded in the Al-Qassim region, Saudi Arabia, as shown in the map in Figure 1, were collected and distributed as follows: (1) Twelve samples were collected from several local farms in the Al-Qassim region; one of these samples was organic wheat. (2) Six samples were purchased from Al-Qassim markets; one of them was organic wheat.

The wheat grains were sifted to remove dirt and dust and checked for visible or indoor contamination. An IKA analytical mill (Staufen, Baden-Wurttemberg, Germany) was used to grind the wheat grain samples into flour. Reagent grade nitric acid (65%) and hydrogen peroxide (33% (w/v)) (Sigma-Aldrich, Germany) were employed for wheat flour digestion. Verification of the accuracy and precision of the proposed method was performed using the certified standard reference material (SRM 1567b) wheat flour obtained from the National Institute of Standards and Technology (NIST, USA). Calibration solutions were prepared daily by serial dilution of a 10 mg/L stock solution of ICP multielement standard solution containing Mg, K, Ca, Al, Mn, Fe, Cu, Zn, Se, Rb, Cd, V, and As (Agilent Technologies, Palo Alto, CA, USA). Standard solutions ranging from 0.1 to 150 μg/L were used for the ICP-MS instrument calibration. Argon with a purity of 99.9999% (White Martins, Saudi Arabia) was used for plasma, nebulization, and auxiliary gas in ICP-MS. Laboratory glassware was kept overnight in 10% (v/v) nitric acid solution. Before use, the glassware was cleaned with ultrapure water and dried in a dust-free atmosphere.

2.3. Digestion Procedures

A 3 : 1 mixture of 8 mol/L nitric acid and 33% hydrogen peroxide was prepared. Next, a 0.3 g–0.5 g portion of the SRM sample, along with an 8 mL–12 mL aliquot of the nitric/peroxide mixture, was added to the PTFE-closed vessel. Analytical blanks were prepared in the same way, in the absence of the sample.

The heating program was performed in five successive steps. In the first step, the temperature was linearly increased to 120°C for 10 min; in the second step, the temperature was linearly increased to 150°C for 10 min; the third step consisted of increasing the temperature linearly to 185°C for 13 min; the fourth step consisted of increasing the temperature linearly to 200 or 210°C for 12 min; in the fifth step, the temperature was kept stable at 200 or 210°C for 15 min. In all steps, the oven was operated at 1100 W.

After the end of the heating program, the vessels were cooled down to room temperature and opened carefully since they might still be pressurized. After sample digestion, the digests were filtered using a 20−25 μm 110 mm diameter filter (Whatman) and transferred to glass volumetric flasks, and the volumes were made up to 50.0 mL with ultrapure water. The samples were then transferred to polypropylene vials.

A two-level factorial experimental design with four variables (24) was employed to optimize the microwave-assisted digestion conditions. The factors were an amount of the sample (0.3–0.5 g), volume of mixture of nitric acid with hydrogen peroxide (8–12 mL), hold time (15–20 min), and temperature (200–210°C). Multiple response (MR) was used in the factorial design, established using the average of the sum of the normalized responses for each analyte and considering their highest value [28]. Table 2 shows the experimental design and MR factors.

2.4. Quality Control and Statistical Analysis

Quality performance parameters, such as accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ), were validated according to validation guidelines [29]. The recovery % was calculated by comparing the determined values with the SRM-certified values three times per day, and precision (repeatability) was obtained from the relative standard deviation (RSD%) of SRM triplicate samples. The LOD and LOQ were calculated as 3 and 10 times σ/S, respectively, where σ is the residual standard deviation of linear regression and S is the slope of the calibration line. The significant difference () between the certified values and the determined values of the SRM was evaluated using the t-test. Other significant differences () in the mean elemental concentrations of the wheat flour samples were assessed using one-way analysis of variance (ANOVA). Statistical analyses were performed using Minitab18 and SPSS 17 software.

3. Results and Discussion

3.1. Optimization of the Microwave-Assisted Digestion Procedure

At temperatures less than 190°C, we observed that the solutions resulting from digestion were yellowish in color with suspended solid particles. Other studies have reported the same observation [30, 31]. At temperatures above 215°C, we observed explosive conditions inside the microwave equipment, probably because of H2O2 decomposition [32]. Using the parameters listed in Table 2, digestion proceeded safely, and all digests were colorless without suspended solid particles.

The MR values obtained for the different experiments are presented in Table 2. The results obtained for the principal effects and interactions are illustrated in the Pareto chart shown in Figure 2.

The use of the 24 factorial design revealed that the only variable, which significantly influenced the digestion of the wheat flour sample, was the temperature, as reflected in the MR data; no statistically significant effects (at the 95% confidence level) were observed for the interactions between the variables.

Figure 3 shows an increase in recovery for all elements when the temperature was raised from 200 to 210°C, keeping all the other variables fixed (refer to experiments 7 and 15 in Table 2).

This effect is also evident from an increase in the MR values from 6.510 to 9.728 at the same temperatures. The value from the ANOVA for the transformed response confirmed statistical significance (). Therefore, the optimum conditions for digestion of the wheat flour samples were determined via factorial design experiments to be those used in experiment 15−0.5 g of the sample and 12 mL of digest solvent mixture, with a microwave-assisted digestion temperature of 210°C and a hold time of 15 min.

The accuracy of the microwave digestion procedure was investigated by applying the method to the standard reference material (SRM 1567b, wheat flour). The measured results obtained using the optimized microwave digestion procedure were in agreement with the certified values for the SRM. This is an indication of the effectiveness of the microwave digestion method as well as the accuracy of element quantification after digestion (t-test, 95% confidence level). The LOD and LOQ values obtained for the proposed method ranged from 0.0001 to 0.63 µg/kg and from 0.0003 to 2.07 µg/kg, respectively, for different elements in wheat flour. The recoveries ranged from 90.43% (Zn) to 109.76% (V), and the precision ranged from 0.42% (K) to 4.89% (Al). The accuracy and precision tests were valid in accordance with the AOAC official methods of analysis [29]. The results are presented in Table 3.

3.2. Application in Wheat Flour Samples

The proposed method was applied to the determination of the macroelements, microelements, and trace toxic elements (Mg, K, Ca, Al, Mn, Fe, Cu, Zn, Se, Rb, Cd, V, and As) in 12 samples of wheat produced in several provinces of the Al-Qassim region of Saudi Arabia and in 6 imported samples of wheat purchased in the Al-Qassim region. Tables 4 and 5 show the element concentrations, the ranges (minimum and maximum values), the mean, and the margin between the minimum and maximum concentrations of elements in the analyzed samples.

High concentrations of K were found in all analyzed samples, ranging from 4433 to 6063 mg/kg in the Al-Qassim samples and from 2042 to 8016 mg/kg in the imported samples. Moderate concentrations of Mg were also found, ranging from 1775 to 2536 mg/kg in the Al-Qassim samples and from 455 to 3466 mg/kg in the imported samples. The average Ca concentration in Al-Qassim and imported samples was 638.5 and 498.5 mg/kg, respectively. The average values for K, Ca, Mg, Zn, Cu, Al, Mn, Se, and V in the Al-Qassim samples were greater than their concentrations in the imported samples. The total concentration of these elements in Al-Qassim samples is 12.55% higher than that in the imported samples. In contrast, the total concentration of Fe, Rb, and Cd in the imported samples is 25.56% higher than that in the Al-Qassim samples. The total concentration of all analyzed elements was 12.41% higher in the Al-Qassim samples than in the imported samples. This means that wheat grown in desert areas and irrigated with high-salinity desert groundwater contains more salt than imported wheat irrigated with fresh water.

3.3. Comparison of Al-Qassim Samples Results with Other Reported Results
3.3.1. Macroelements: Potassium, Magnesium, and Calcium

Table 6 shows the results for potassium, magnesium, and calcium in whole wheat samples reported by other researchers and those obtained in the present study. The mean levels of the macroelements in all samples of Al-Qassim wheat flour are markedly higher for magnesium and slightly higher for calcium and potassium compared to the data reported by the other authors. The sum of the mean concentrations of macroelements in Al-Qassim wheat flour samples is 34.22% higher than the sum of the mean concentrations reported by the authors under comparison.

3.3.2. Microelements: Manganese, Iron, Copper, and Zinc

A comparison between the results for the microelements obtained in this study and those of other authors can also be seen in Table 6. In the case of manganese, the mean concentrations determined in the Al-Qassim samples are lower than those published by Vrček and Vinković Vrček [8] and Ficco et al. [34] and higher than those of the other authors [26, 33, 35]. The mean iron concentration in Al-Qassim samples is lower than that recorded in samples from Italy, Croatia, and Latvia and higher than the data recorded in Brazil and China. The mean concentrations determined for Cu and Zn in the Al-Qassim samples are higher than those published by all the authors under comparison, except for the lower zinc data published by Vrček and Vinković Vrček [8]. The sum of the mean concentrations of microelements in Al-Qassim wheat flour samples is 11.66% higher than the sum of the mean concentrations reported by the authors under comparison. The total amount of macroelements and microelements under the study in the Al-Qassim samples is 33.90% higher than the comparison samples. The high quality of Qassim wheat is consistent with it being a food source of minerals, as the results and comparison indicate the high percentage of minerals in the Qassim samples compared to the imported samples and the samples under comparison.

3.4. Multivariate Analysis Results
3.4.1. Hierarchical Cluster Analysis (HCA)

Hierarchical cluster analysis was performed for all elements studied in the Al-Qassim wheat samples. Hierarchical cluster analysis was applied to the autoscaled data using the complete linkage method with Euclidean distances to calculate the sample interpoint distances and similarities. The smallest distance indicated the highest degree of the relationship; therefore, those objects are considered to belong to the same group. All samples from different regions were separated into two clusters based on the dendrogram cut at a distance of 15 (Figure 4). Six samples were grouped in each cluster.

The first cluster was composed of samples from Al Bukayriah: Al Bukayriah and Al_fowileq (n = 2), Al Badaye: Al Badaye (n = 1), Al Rass: Al Rass and Maragan (n = 2), and Uyon Al Jiwa: Solobiah (n = 1).

The second cluster was composed of samples from Al Bukayriah: Al Shihiah (n = 1), Riyadh Al-khabra: Riyadh Al-khabra (n = 1), Al Rass: Al Fawarah (n = 1), Al Mithnab: Al Mithnab (n = 1), and Buraydah: Buraydah and Al_qara’a (n = 2).

The results indicated that HCA analysis could indicate an approximate location but could not determine the geographical origin of wheat flour.

3.4.2. Principal Component Analysis

The principal component (PC) loading plot for three PCs of metals and the principal component score plot of spices (PC1 × PC2) are illustrated in Figures 5(a) and 5(b).

The principal elements used to discriminate between wheat flour samples produced in seven different provinces of the Al-Qassim region in Saudi Arabia were further investigated. The results indicate that there were five eigenvalues higher than 1.00, and the three PCs explain 70.71% of the total variance (Table 7). The contribution of the first factor was 34.56%, which shows high loadings for Al, V, Fe, Cu, Zn, and As and a weak loading for Se (0.459). The second component explains 20.73% of the total variance. This component showed high loadings for Mg, Mn, Cd, and Se, with a weak loading for Fe (0.414). The third component explaining 15.41% of the total variance includes high loadings for Ca and Mn, with a weak loading for Cd (0.386).

4. Conclusion

A microwave digestion method using a mixture of dilute nitric acid and hydrogen peroxide was optimized; suitable conditions for the digestion of wheat flour samples were achieved and used for the determination of the macroelements, microelements, and toxic elements. The validity of the analytical method was proven, and the accuracy was confirmed via analysis of a certified reference material consisting of wheat flour. The results from analyzing samples of wheat produced in the Al-Qassim region clearly showed variations in essential minerals compared to data for wheat flour produced in several other countries as well as to brands of imported wheat flour sourced from Al-Qassim markets. The PCA and HCA models were efficient for the classification of Al-Qassim wheat samples, providing useful information on similarities between the samples.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors gratefully acknowledge Qassim University, represented by the Deanship of “ Scientific Research, on the financial support for this research (grant no. COS-2022-1-1-J-24975) during the academic year 1444 AH/2022 AD.”