Development and Application of an ICP-AES Method for the Determination of Nutrient and Toxic Elements in Savory Snack Products after Autoclave Dissolution

: In this study, a method for the determination of trace elements in snacks using inductively coupled plasma–atomic emission spectrometry (ICP-AES) is presented. The examined elements were Pb, Ni, Cr, Cu, Mg, Zn, Fe, Al, Ba, Ca, Co, Mn and Cd. Under the optimized conditions, digestion of 300 mg of the snack samples was performed by the addition of 5 mL of nitric acid in a Teﬂon autoclave and by heating the obtained mixture at 120 ◦ C for 75 min. In order to evaluate the e ﬃ ciency of the proposed protocol, method linearity, accuracy precision, limits of detection (LODs) and limits of quantiﬁcation (LOQs) were evaluated. The relative standard deviations (RSD%) for all elements were lower than 13.5%, demonstrating that the method o ﬀ ered good precision. The relative recoveries values (R%) ranged between 80–120%, demonstrating that the method o ﬀ ered good accuracy. The LODs for all the trace elements ranged between 0.18 and 3.75 µ g g − 1 , while the LOQs ranged between 0.60 and 12.50 µ g g − 1 . Finally, the proposed protocol was implemented for the analysis of a wide variety of savory snack samples including commercial snacks from corn, potato chips, popcorns, pu ﬀ ed rice cake and crackers.


Introduction
Elemental food composition data are important to consumers and health professionals since metals play a significant role in human nutrition, because of their toxic and carcinogenic effects [1]. In foodstuffs, the major chemical elements are sodium, calcium, potassium and magnesium [2]. Other nutrient elements that are considered essential for human health include copper, iron and zinc [3]. Metals such as barium are generally considered non-essential and their function in biological systems is not known [4]. Finally, there are elements like lead, cadmium and mercury that exhibit high toxicity, and they occur in the environment mainly due to environmental pollution from anthropogenic sources [5]. These metals can migrate through the food chain to humans. As a result, it is critical to determine them in foodstuffs.
There are multiple potential sources of mineral occurrence in food samples, including their initial presence in the raw materials, the processing practices used for food production, contamination from the equipment used during production and/or storage and contamination of the environment. Toxic chemical elements primarily enter foodstuffs due to the contact with the environment such as polluted soil, water and air that falls as precipitation with rain and can contaminate food raw materials [6,7].
Snacks like potato chips, corn snacks and popcorns are common in daily diet, while they are widely consumed in large amounts also from children [8]. Potato chips can be produced by deep fat frying in order to have a unique texture flavor combination, that makes them desirable for the consumers. Various parameters including nature of oil, temperature and duration of frying, and storage conditions can determine the quality of chips [9]. Popcorn (Zea mays Everta) is another popular snack product being considered healthier than chips due to its higher fiber and antioxidant content [10]. Upon heating to sufficient temperature, the caryopsis expands resulting in a large expansion of volume, producing the desired product [11]. Crackers are also popular snacks with low contents of moisture, sugar and fat that exhibit a thin and crispy texture. Crackers are also widely consumed worldwide [8].
Among the several methods for the determination of trace elements, atomic spectroscopy techniques are considered the most sensitive, selective and rapid. The most widely used spectroscopic techniques for the qualitative and quantitative determination of metals in foodstuffs include inductively coupled plasma atomic emission spectroscopy (ICP-AES) [12], inductively coupled plasma mass spectrometry (ICP-MS) [13] flame atomic absorption spectrometry (FAAS) [14], cold vapor atomic absorption spectroscopy (CV-AAS) [15] and electrothermal atomic absorption spectroscopy (ETAAS) [16]. ICP-AES exhibits many benefits including high sensitivity, fastness, simplicity of analysis and ease in overcoming interferences [12].
For the determination of trace elements in food samples by ICP-AES, the sample has to be converted into a solution. Therefore, sample digestion procedure is necessary prior to sample analysis [17]. Multiple digestion techniques including dry ashing and wet ashing, as well as conventionally heating procedures, microwave digestion and acid bomb digestion have been studied. Wet digestion is an important methodology for the digestion of the trace heavy metals in solid samples. Nitric acid is an important oxidizing acid that can be employed for wet digestion of foodstuffs that have a high carbohydrate and fat content, in order to reduce carbonization and to ensure the efficiency of the digestion process [4].
In this study, a simple and rapid ICP-AES method was developed for the determination of trace elements (Pb, Ni, Cr, Cu, Mg, Zn, Fe, Al, Ba, Ca, Co, Mn and Cd) in savory snack samples (i.e., corn snacks, potato chips, popcorns and crackers) after wet digestion with nitric acid. Various dissolution procedures were investigated in order to achieve complete digestion of the examined samples. Furthermore, validation of the proposed method was performed in terms of linearity, limits of detection, limits of quantification, accuracy and precision. The developed method was successfully employed for the analysis of savory snacks of different types from Greek market.

Materials and Chemicals
Nitric acid 65% of analytical grade was supplied by Merck (Darmstadt, Germany) and used for sample digestion and solution preparation. High purity double distilled water was used in all the experiments. Stock standard solutions (1000 mg L −1 ) of lead, nickel, chromium, copper, magnesium, zinc, iron, aluminum, barium, calcium, cobalt, manganese and cadmium in 0.5 M nitric acid were supplied by Merck (Darmstadt, Germany). Working standard solutions were prepared daily by appropriate serial dilutions of the stock solutions in 0.5 M nitric acid.

Instrumentation
In this study, a Perkin-Elmer Optima 3100XL axial viewing ICP-AES equipped with a cyclonic spray chamber and a GemTip cross-flow nebulizer was used for the determination of the trace elements. The samples were introduced into the ICP-AES system at a flow rate of 1.0 mL min −1 , using a three-channel peristaltic pump and Tygon-type polyvinyl chloride (PVC) peristaltic pump tubes (inner diameter 0.030 in). The nebulizer argon gas flow rate, the auxiliary argon gas flow rate and the plasma argon gas flow rate were 0.85, 0.6 and 15 L min −1 , respectively. Nine points per peak were used for the measurement of the peak area. The operating conditions of the ICP-AES system were as such: power output: 1.5 kW, pixel resolution: 0.006 nm at 200 nm, torch type: Fassel type, fully demountable and torch alumina injector inner diameter: 2.0 mm. Two analytical wavelengths were recorded for each element and the peak area was chosen as a signal measurement mode. Table 1 summarizes the recorded emission lines for each element. Digestion of the snack samples was performed in Teflon ® (DuPont, DE, USA) vessels. The vessels were placed in a steel autoclave and heated in a six-position aluminum block (Berghof, BTR, 941, Eningen, Germany). The maximum working volume of the vessels were 50 mL, and the maximum operating temperature was 180 • C.
All digestion vessels, glassware and storage bottles were soaked in 10% (v/v) nitric acid for at least 24 h and they were washed extensively with double de-ionized water prior to their use.

Sample Collection
In this work twenty-one snack samples (i.e., nine snacks from corn coded as SFC, three popcorn samples coded as PC, one puffed rice cake sample coded as PRC, six potato chips samples coded as P and two cracker samples coded as CR) were analyzed to determine the concentrations of Pb, Ni, Cr, Cu, Mg, Zn, Fe, Al, Ba, Ca, Co, Mn and Cd. The snacks analyzed in this study belong to the most popular brands in Greek market. The following snacks from corn were analyzed: SFC-1 (cheese flavor, oven baked corn snack), SFC-2 (pizza flavor, oven baked corn snack), SFC-3 (cheese flavor, oven baked corn snack) , SFC-4 (cheese flavor, oven baked corn snack), SFC-5 (cheese and tomato flavor, oven baked corn snack), SFC-6 (cheese flavor, oven baked corn snack), SFC-7 (oven baked corn snack with peanut and chestnut), SFC-8 (oven baked corn snack with peanut and chestnut) and SFC-9 (bacon flavor, oven baked corn snack). The following popcorn samples from corn were analyzed: PC-1 (popcorn with salt), PC-2 (butter flavor, popcorn sample) and PC-3 (popcorn with salt). The following puffed rice cake sample was analyzed: PRC-1 (oregano flavored puffed rice cakes). The following potato chip samples were analyzed: P-1 (potato chips with salt), P-2 (potato chips with salt), P-3 (potato chips with salt), P-4 (potato chips with oregano flavor), P-5 (potato chips with oregano flavor) and P-6 (potato chips with barbeque flavor). The following cracker samples were analyzed: CR-1 (crackers with salt) and CR-2 (crackers with salt). All samples were purchased from local shops in Thessaloniki, Greece.

Validation of the ICP-AES Method
The herein reported ICP-AES method was validated in terms of linearity, limits of detection (LOD), limits of quantification (LOQ), accuracy and precision.
For the evaluation of method linearity, calibration curves for each element were constructed by plotting the peak area of the optimum emission line to the concentration of the standard solution solutions or spike solutions for standard addition curves. Subsequently, least square linear regression analysis was used to evaluate the slope, intercept and coefficient of determination.
The method's accuracy was evaluated in terms of bias between nominal and measured concentrations of spiked samples, expressed as relative recovery (R%) and the method precision, expressed as relative standard deviation (RSD%), was evaluated by analyzing in five replicate measurements spiked sample solutions.
Finally, for the LODs and LOQs, ten separate blank solutions were independently prepared and analyzed. The LOD value was the concentration that corresponds to three times the standard deviation of the measurements for the blank solutions divided by the slope of calibration curves for each element, while the LOQ value was the concentration that corresponds to ten times the standard deviation of the measurements for the blank solutions divided by the slope of calibration curves for each element [18,19].

Selection of the Emission Line
The selection of the optimum emission lines was based on the intensity and their sensitivity (i.e., the higher slope among the calibration curves for the recorded emission lines), as well as the absence of spectral interferences.

Sample Preparation of the Snack Samples
In order to find the optimal digestion procedure for complete sample decomposition, different wet acid methods were tested. For this purpose, the sample mass was kept constant and different digestion mixtures were evaluated. The final digest was diluted to a final volume of 25 mL in volumetric flasks with double-deionized water. Table 2 summarizes the observations on the effectiveness of the dissolution tests. Dissolution trials were performed with 300 mg of sample (SFC-1, PC-1, PRC-1, P-1 and CR-1). Further increase of the sample amount resulted in slight sedimentation and it probably required higher consumption of nitric acid. Therefore, 300 mg of sample were chosen to avoid high consumption of sample and chemicals. As it can be observed, in order to obtain a clear sample solution without precipitation, autoclave digestion of 300 mg of sample with 5 mL of nitric acid for 75 min at 120 • C gave the best results. Finally, these conditions were chosen for snack samples digestion. Alternative, digestion time can be further reduced with the use of microwave assisted digestion.

Figures of Merit
The performance of the ICP-AES method was evaluated under the conditions described above. Table 3 shows the calibration curve, the linear range and the LOD and LOQ for each element. As it can be seen, coefficients of determination for all metals were very good (r 2 > 0.9998) and wide linear ranges were obtained. According to the specifications of the International Union of Pure and Applied Chemistry (IUPAC) [20], blank solutions were used to calculate the LOD and LOQ values of the method. The LODs and LOQs of the ICP-AES method ranged between 0.18-3.75 µg g −1 and 0.60-12.5 µg g −1 , respectively. In order to evaluate the accuracy and the precision of the ICP-AES method, spiked standard solutions at two different concentration levels (50 and 100 µg g −1 ) were analyzed 5 times. Tables 4 and 5 summarize the accuracy and precision results for the examined elements. The relative recovery in the spiked samples ranged between 80-120% for all concentration levels, indicating that the accuracy of the ICP-AES method is satisfactory. Finally, the RSD% values were lower than 13.5%, indicating that the precision of the ICP-AES method is satisfactory.
The herein developed method was compared with other studies reported in the literature. Gopalani et al. [17] performed wet digestions of potato chips and biscuits for the determination of their content in various metals (i.e., aluminum, cadmium, cobalt, chromium, copper, iron, manganese, magnesium, nickel, lead and zinc). For sample dissolution, wet digestion of 1 g of sample was performed with three different mixtures of acids i.e., HNO 3 :H 2 O 2 (8:4), HNO 3 :H 2 SO 4 (8:4) and HNO 3 :HCl (8:4). In total, 12 mL of reagents were required, while dissolution was achieved at 130 • C in 3 h. Narin et al. [21] performed wet digestions of potato and corn chips for the determination of iron, zinc, manganese and copper. Dissolution of 1 g of the sample was achieved by the addition of a mixture of HNO 3 :H 2 O 2 (8:4). In this case, the duration of the wet digestion was also 3 h at 130 • C, while it required 12 mL of the dissolution mixture. It can be concluded that the employment of autoclave dissolution can significantly reduce the requirements for sample and reagent consumption, while it also reduce the sample preparation time. It is important to mention that microwave digestion can be employed to further reduce the total analysis time.

Analysis of Snack Products
The results from the analysis of various snack samples collected from the Greek market are presented in Table 6. All samples were analyzed in duplicate. As it can be observed, magnesium and calcium were the major elements in snack samples since their concentrations were between 242.5-1556.3 µg g −1 and 31.0-767.4 µg g −1 , respectively. Iron, copper, zinc and aluminum were detected in most samples and their concentrations were between 5.9-305.0 µg g −1 for Fe, 3.3-7.7 µg g −1 for Cu, 3.3-19.4 µg g −1 for Zn and 4.6-54.2 µg g −1 for Al. Chromium, nickel, manganese and barium were also present in the snack samples. Their concentrations were between 0.60-1.26 µg g −1 for Cr, 0.91-2.1 µg g −1 for Ni, 1.9-6.0 µg g −1 for Mn and 0.83-2.30 µg g −1 for Ba. Finally, the concentrations for lead, cadmium and cobalt were lower than the method LOD. 45.

Conclusions
In this work, a simple and rapid ICP-AES method was developed and applied for the determination of major, minor and trace elements (Pb, Ni, Cr, Cu, Mg, Zn, Fe, Al, Ba, Ca, Co, Mn and Cd) in snack samples. Various wet digestion procedures with nitric acid were evaluated for the complete dissolution of the snack samples. It was observed that 5 mL of nitric acid was required for the digestion of 300 mg of snack samples at 120 • C, in a steel autoclave with Teflon vessels. After, optimization of the digestion procedure of the snack samples, the proposed method was validated in terms of linearity, LOD and LOQ values, accuracy and precision. Finally, twenty-one snack samples (i.e., potato chips, snacks from corn, popcorns, puffed rice cake and crackers) from the local market were analyzed. Calcium and magnesium were the major elements in the samples. Nickel, chromium, coper, manganese, zinc, iron, aluminum and barium were also quantitatively determined in most samples. Finally, lead, cadmium and cobalt were not detected in the samples. The proposed method is likely to be applicable to the analysis of other similar complex food products.