TX_1~ABS:AT/ADD:TX_2~ABS:AT 26 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2022, 6 (1): 26-31 ReseaRch aRticle Determination of Elements in Indigenous Vegetables Using ICP-MS Bashdar A. Sadee1,2 1Department of Food Technology, College of Agricultural Engineering Sciences, Salahaddin University-Erbil, Kurdistan Region-Iraq, 2Department of Nutrition and Dietetics, Cihan University-Erbil, Kurdistan Region, Iraq ABSTRACT Determination of elements in indigenous vegetables is important as source of dietary that are essential for human consumption. Heavy metal concentrations were determined in indigenous vegetables collected in Erbil city-Kurdistan region of Iraq. ICP-MS was applied to determine total concentrations of Al, Co, Fe, Mn, Ni, Se and Zn in Malva parviflora, Gundelia tournefortii, Arum spp, Spinacia oleracea, Mentha longifolia, Beta vulgaris subsp, Apium graveolens, Lepidium sativum L, Allium Kurrat. Schweinf and Allium fistulosum after acid digestion of the samples with the aid of microwave using nitric acid/hydrogen peroxide. The contents of heavy metals in vegetable samples varied between 40 and 451 for Al, 0.04 and 0.96 for Co, 53 and 247 for Fe, 3.37 and 11.58 for Mn, 0.26 and 5.53 for Ni, 0.09 and 0.98 for Se and 1.58 and 4.41 for Zn on the basis of dry weight. The methodology was validated by certified reference material GBW10015-spinach and inductively coupled plasma mass spectrometry analysis. The levels of Al, Mn, and Ni in some investigated vegetables are higher than the permissible limits for human consumption. Keywords: Heavy metals, ICP-MS, indigenous vegetables, microwave acid digestion, validation INTRODUCTION Recently, more attention has been paid to cumulating of heavy metals and metalloids in vegetables. Vegetables can be exposed to environmental pollution more than other food systems because of loading vegetables into the air. The accumulation and taken up of heavy metals by vegetables in eatable and non-eatable fractions at a certain quantity may lead to health concerns to both living organisms and humans.[1] Food safety concerns may occur because of phytotoxicity and absorption of high levels of heavy metal by vegetation resulted from transferring high levels of these heavy metals from agriculture soils.[2,3] Bioaccumulation high quantity of toxic heavy metals in vegetables can cause the deficiency of nutrients of dietary to human beings or may cause health issues for both human beings and ecosystem.[4-7] Heavy metals can be categorized into vital metals (Fe, Mn, Cu, Se, Zn and etc.), possibly essential (Ni, V, and Co) and conceivably toxic (As, Cd, Pb, Hg, etc.). Essential metals play a major role for living organisms if they exist at low or high quantity, while exposures to toxic metals may harm even at low level for long-term exposure. Exposure to toxic metals can cause various serious health issues with changing degree of harshness and circumstances such as issues of kidney, neurobehavioral and developmental disarrange, hypertension, and probably cancer.[8-13] Furthermore, numerous of nervous, cardiovascular, renal, neurological deterioration in addition bone illness and many other health issues may occur because of high content exposure of metals beyond Maximum Permissible Level (MPL).[1,14] Humans consume vegetables in both cooked and raw forms because they are considered as an essential part of their diet. Vegetables function as acid generation throughout digestion and some available metals in vegetables are even vital biochemically and psychologically from health prospective.[15] Although Al is relatively low bioavailable, it has been suggested that there is a connection between aluminum and Alzheimer’s disease.[16] Human metabolism can be regulated via metals including, Co, Fe, Mn, Se, and Zn. Mn is one of the important components of many enzymes exist in human and considered an essential element functions as an activator. Although Co and Ni are vital for human beings, higher levels than the recommended values lead to metabolic abnormal.[1] This study was conducted to evaluate the contents of heavy metals found in various edible indigenous vegetables. Corresponding Author: Bashdar A. Sadee, Department of Nutrition and Dietetics, Cihan University-Erbil, Kurdistan Region, Iraq. E-mail: bashdar.sadee@su.edu.krd Received: December 26, 2021 Accepted: February 16, 2022 Published: March 6, 2022 DOI: 10.24086/cuesj.v6n1y2022.pp26-31 Copyright © 2022 Bashdar A. Sadee. This is an open-access article distributed under the Creative Commons Attribution License (CC BY-NC-ND 4.0). Cihan University-Erbil Scientific Journal (CUESJ) Cihan University-Erbil Scientific Journal (CUESJ) Sadee: Determination of elements in indigenous vegetables using ICP-MS 27 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2022, 6 (1): 26-31 EXPERIMENTAL Chemicals and Reagents All chemical compounds utilized in this work were of high purity reagent and Milli-Q water (18MΩcm) was used to prepare all solutions. A standards stock solution of concentration 100 µg mL−1 in 5% Nitric acid (HNO 3 ) for Al, Co, Fe, Mn, Ni, Se, and Zn was obtained from CPI international, USA. HNO 3 70% reagent was ordered from Merck (Germany) and used to digest vegetable samples to obtain extracts of Al, Co, Fe, Mn, Ni, Se, and Zn. Hydrogen peroxide (H 2 O 2 ) 37% was purchased from Fischer Scientific, United Kingdom. A certified reference material (Spinach GBW10015) was supplied by the Institute of Geophysical and Geochemical Exploration, Langfang, China. All glasswares used in this work then rinsed with Milli-Q water followed by soaking it in HNO 3 acid before use. Instrumentation Inductively coupled plasma mass spectrometry (ICP-MS, Perkin-Elmer SCIEX ELAN 6100 DRC II) was applied to identify the total elements (Al, Co, Fe, Mn, Ni, Se, and Zn) in the analyzed vegetable samples. The instrument was supplied with a Sturman-masters spray chamber and a V-groove quartz concentric nebulizer for sample induction. In concise, the optimum parameters of the instrument were a Radio frequency power of 1250 W with Ar flow rates of 13, 0.8 and 0.95 L min-1 for cool, auxiliary, and sample gas, respectively. For the experimental results, the intensity of the following isotopes was measured: 55Mn, 59Co, 27Al, 56Fe, 60Ni, 66Zn, and 78Se. Possible interferences were eliminated using collision cell technology and also 7 % H 2 in He gaseous with average flow rates of 3.4 ml. min−1 was used. Internal standards such as Indium (In) and iridium (Ir) were used for all vegetable samples at 10 µg L−1 (final concentration). Sampling and Sample Preparation A vegetable samples were purchased from the local markets located in Erbil, Kurdistan Region of Iraq in 2021. The common and scientific names of analyzed vegetables are presented in Table 1. All samples were rinsed with Milli-Q water and placed in a sealed plastic box. A freeze drier was used to dry all vegetable samples for 48 h at −40°C. A fine powder of vegetable samples with 180 µm size obtained by sieving of grounded samples using agate pestle and mortar. Determination of Total Metals in Vegetable Samples 0.2g of freeze-dried vegetable samples was weighted in Teflon vessels with Teflon covers, 5 mL HNO 3 ( 70%) and 2 mL H 2 O 2 (30%) were added. The vegetable samples were acid digested using MARS X-press (CEM, USA) microwave using a previous method by Sadee et al.[17] The applied digestion program was as follows: vegetable samples were digested using the microwave for 43 min at 1600 W. Initially, the temperature was raised up to 160°C in 15 min and stayed at this temperature for 5 min. Then, the temperature ramped from 160 to 200°C over 8 min and stayed at this temperature for 15 min. The Teflon vessels were left to cool at room temperature. After digestion, vegetable samples were transferred into a volumetric flask (25 mL capacity), fortified with internal standards of In and Ir with an ultimate contents of 10 µg L−1 and made up to final volume (25 mL) with 2% (v/v) HNO 3 using Milli-Q water. Total metals in selected vegetables were determined utilizing ICP-MS. GBW10015-spinach certified reference (CRM) material was applied validation of the method. RESULTS AND DISCUSSION Figures of Merit The slop of the applied calibration curve was used to calculate the detection limit (DL) of the ICP-MS instrument (μg g−1) and 3 times standard deviation of 10 measurements of blank value. The results of DL quantities of the ICP-MS are presented in Table 2. Validation of Analytical Method In order to check the quality control of used procedures, total Al, Co, Fe, Mn, Ni, Se, and Zn were quantified in GBW10015- spinach. The results for, total Al, Co, Fe, Mn, Ni, Se, and Zn contents in the CRM are tabulated in Table 3. The elemental contents resulted from the experiment were in well agreement with the certified quantities (the recovery of examined elements ranged from 94% to 105%). Table 1: Vegetables English common name and scientific name of collected samples Common English name Scientific name Mallow Malva parviflora Sunflower Gundelia tournefortii Arum Arum spp Spinach Spinacia oleracea Wild mint Mentha longifolia Chard Beta vulgaris subsp Celery Apium graveolens Garden cress Lepidium sativum L Leek Allium Kurrat Schweinf Spring onion Allium fistulosum Table 2: Limit of detection for Al, Co, Fe, Mn, Ni, and Se (μg g−1 dry weight) measured as the mean+3 SD of the signal of method blank Metals Concentration μg g−1 Al 0.03 Co 0.03 Fe 0.02 Mn 0.02 Ni 0.03 Se 0.05 Zn 0.02 Sadee: Determination of elements in indigenous vegetables using ICP-MS 28 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2022, 6 (1): 26-31 Total Metal Determinations in Vegetables The averaged results of heavy metal concentrations in selected varieties of indigenous vegetables based on dry weight are shown in Table 4 and the literature reported values from various origin of the world from corresponding vegetables are listed in Table 5. Aluminium The concentration of Al in this study ranged from 40 to 451 µg g−1 [Table 4]. The lowest concentration of Al in analyzed vegetables was found in Malva parviflora with 40 µg g−1, while the maximum level of Al was detected in Spinacia oleracea with value of 451 µg g−1. It was noticed that the Al contents in the vegetable samples collected were in increasing order of M. parviflora > Lepidium sativum L > Mentha longifolia > Apium graveolens > Allium Kurrat Schweinf > Beta vulgaris subsp > Gundelia tournefortii > Arum spp > Allium fistulosum > S. oleracea. The observed Al concentration in vegetable samples is compared to the literature reported values ranged 0.160–131.640 µg g−1 [Table 5]. The values of Al content of each investigated vegetable of the current study higher than that reported for the corresponding vegetables by other researchers. The concentration of Al in vegetables of this study except M. parviflora is higher than the maximum permissible dose limit recommended for Al, 60 mg/day.[18,19] Cobalt The concentrations of Co in examined vegetables of this study ranged between 0.040 and 0.143 µg g−1 [Table 4]. Although the concentration of Co in vegetables of M. parviflora, G. tournefortii and L. sativum L. were below the limit of detection of the method, the highest Co content was found in A. fistulosum which was 0.143 µg g−1. The Co levels in the vegetable samples collected were in increasing order of M. parviflora ≈ G. tournefortii ≈ L. sativum L > A. Kurrat Schweinf > A. graveolens > Arum spp > B. vulgaris subsp > S. oleracea > M. longifolia > A. fistulosum. The observed literature values of Co were in the range of 0.01 - 3.00 µg g−1 [Table 5]. The values obtained for Co vegetables in the current study are lower than that reported in the literature for the same vegetable species except M. longifolia, A. graveolens and A. fistulosum. The concentration of Co in the vegetables of this study is well within MPL of Co for adults, 50 µg/day.[8,19] Iron The quantities of Fe varied according to the types of vegetables concentred with quantities between 53 and 247 µg g−1 [Table 4]. The minimum and maximum Fe contents of the samples were found to be 53 µg g−1 in B. vulgaris subsp and 247 µg g−1 in S. oleracea, respectively. The Fe concentrations in the vegetable samples collected were in increasing order Table 3: total metal concentration of CRM GBW10015-spinach; all experimental values are calculated in μg g−1, mean±standard deviation (n=3) Metals Certified value Experimental value obtained Extraction efficiency % Al 610±60 602±40 99 Co 0.220±0.03 0.229±0.016 104 Fe 540±20 546±42 101 Mn 41±3 42±0.59 102 Ni 0.920±0.012 0.966±0.020 105 Se 0.092±0.024 0.087±0.004 95 Zn 35.300±1.5 33.450±0.449 95 Table 4: Results of analysis for total metal in the selected vegetables dry weight (mean±standard deviation) (n=3) Vegetable Al μg g−1 ± SD Co μg g−1 ± SD Fe μg g−1 ± SD Mn μg g−1 ± SD Ni μg g−1 ± SD Se μg g−1 ± SD Zn μg g−1 ± SD Malva parviflora 40±0.352 <0.03 58±0.584 4.930±0.044 0.260±0.002 <0.05 4.412±0.030 Gundelia tournefortii 151±1.985 <0.03 100±0.805 3.370±0.003 0.532±0.004 <0.05 3.483±0.028 Arum spp 298±1.640 0.050±0.006 139±0.791 5.330±0.066 1.142±0.007 <0.05 3.092±0.021 Spinacia oleracea 451±5.361 0.960±0.001 247±2.275 9.382±0.099 1.351±0.015 0.090±0.014 4.080±0.035 Mentha longifolia 103±1.102 0.120±0.013 198±3.823 6.731±0.159 1.533±0.019 <0.05 3.691±0.054 Beta vulgaris subsp 140±0.932 0.070±0.002 53±0.341 11.580±0.076 0.564±0.006 0.980±0.031 4.295±0.039 Apium graveolens 124±1.525 0.040±0.001 81±1.020 9.622±0.115 0.812±0.008 <0.05 3.234±0.035 Lepidium sativum L 65±2.191 <0.03 131±0.415 4.183±0.017 0.722±0.004 0.494±0.004 3.195±0.023 Allium Kurrat Schweinf 132±0.633 0.040±0.001 75±0.820 10.891±0.161 0.473±0.005 <0.05 1.588±0.026 Allium fistulosum 334±3.416 0.140±0.003 138±0.305 5.142±0.035 5.533±0.081 0.242±0.015 2.420±0.024 Sadee: Determination of elements in indigenous vegetables using ICP-MS 29 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2022, 6 (1): 26-31 of B. vulgaris subsp > M. parviflora > A. Kurrat Schweinf > A. graveolens > G. tournefortii > L. sativum L > A. fistulosum > Arum spp > M. longifolia > S. oleracea. In the literature Fe concentration has been documented in the range of 28-1952 µg g−1 [Table 5]. The values of Fe varied according to the vegetable species, the concentrations of Fe in this study are lower than the same vegetables reported in the literature except of S. oleracea. The results of the present work with the exception of S. oleracea are well within the Provisional Maximum Tolerable Daily Intake (PMTDI) of Fe is 0.8 mg/kg bw.[20] Manganese The levels of Mn in investigated vegetables of this study ranged between 3.374 and 11.580 µg g−1 [Table 4]. The lowest concentration was seen in G. tournefortii (3.374 µg g−1) sample while the highest level was 11.580 µg g−1 for B. vulgaris subsp. The Fe concentrations in the vegetable samples collected were in increasing order of G. tournefortii > L. sativum L > M. parviflora > A. fistulosum > Arum spp > M. longifolia > S. oleracea > A. graveolens > A. Kurrat Schweinf > B. vulgaris subsp. The reported literature concentrations of Mn varied from 8.4 to 178.53 µg g−1 [Table 5], which are higher than the values obtained for the same vegetables of this investigation. The concentration of Mn in the investigated samples except M. parviflora, G. tournefortii, and L. sativum L is above the recommended intake range of Mn for an adult, 2-5 mg/day.[8] Nickel The calculated concentration of Ni in this study was recognized in the range of 0.264–5.532 µg g−1 [Table 4]. The lowest level of Ni in this study was found in M. parviflora which was 0.264 µg g−1, whereas the highest content of Ni was 5.532 µg g−1 for A. fistulosum. The Ni contents in the vegetable samples collected were in increasing order of M. parviflora > A. Kurrat Schweinf > G. tournefortii > B. vulgaris subsp > L. sativum L > A. graveolens > Arum spp > S. oleracea > M. longifolia > A. fistulosum. The reported literature values of Ni range from 0.1 to 21.1 µg g−1 [Table 5]. The contents of Ni in vegetables under study were higher than those corresponding vegetables documented in the literature except G. tournefortii, S. oleracea, B. vulgaris subsp, and L. sativum L. The concentration of Ni in vegetables of present work is well above the permissible limit of daily intake of Ni (0.1 mg/day), which is alarming.[18,19] Selenium Se was lower than the DL (0.030 µg g−1) in the examined vegetable samples of the current study with the exception for S. oleracea, A. fistulosum, L. sativum L. and B. vulgaris subsp were 0.094, 0.241, 0.490 and 0.980 µg g−1, respectively [Table 4]. The literature values of observed concentrations of Se are varying from 0.020 to 762 µg g−1 [Table 5]. Except A. fistulosum, the results for this study for Se in vegetables under investigation including S. oleracea, A. graveolens, and A. Kurrat Schweinf were lower than the literature values reported Table 5: Heavy metal concentration in various vegetables in different countries (literature values) Vegetable Concentration μg g−1 Location Sampling period Reference Al Co Fe Mn Ni Se Zn Malva parviflora NA NA 296 NA 0.000 NA 59 Ethiopia 2012 [21] Gundelia tournefortii NA NA NA NA NA 1.080 1952 NA 408.370 NA 8.400 178.530 8.400 NA NA NA NA NA 820 NA 20.050 Iran Turkey Turkey Not mentioned 2001 Not mentioned [22] [23] [24] Arum spp NA 131.640 NA 1.4100 NA NA 166.400 NA NA 9.900 NA NA 17.140 NA NA NA NA 762 9.60 NA NA Saudi Arabia Iran Turkey 2003 Not mentioned Not mentioned [25] [26] [27] Spinacia oleracea NA 570 0.01 NA 374.700 NA 131.900 NA 0.000 NA NA 0.177 202.40 NA Iran India 2007 2004 [28] [29] Mentha longifolia NA 3 146.300 30.730 21.100 NA 28.100 Saudi Arabia 2003 [25] Beta vulgaris subsp NA 70 0.010 NA 257.300 NA 91.770 NA 0.000 NA NA 0.020 188.000 NA Iran Finland 2007 2003 [28] [30] Apium graveolens NA 0.330 1678 164.300 3.260 NA 100 Iran 2007 [28] Lepidium sativum L NA 0.160* NA 1.700 NA NA 118.2 NA NA 13.840 NA NA 0.100 NA NA NA NA 2.550 17.400 NA NA Saudi Arabia Sweden Turkey 2003 1992 2014 [25] [31] [32] Allium Kurrat Schweinf 50 0.040 28 14 0.280 0.110 16 Finland 2003 [30] *Wet weight; NA: Not Analysed Sadee: Determination of elements in indigenous vegetables using ICP-MS 30 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2022, 6 (1): 26-31 for the same corresponding vegetables. The content of Se in S. oleracea, B. vulgaris subsp, L. sativum L, and A. fistulosum is higher than the tolerable daily intake of Se which is 55 µg/day.[8] Zinc The concentrations of Zn in vegetables under investigation ranged between 1.588 and 4.295 µg g−1 [Table 4]. The minimum level of Zn in the analyzed vegetables of the current study was detected in A. Kurrat Schweinf which was 1.588 µg g−1, meanwhile the maximum content of Zn in vegetables understudy was detected in B. vulgaris subsp (4.295 µg g−1). The Zn concentrations in the vegetable samples collected were in increasing order of A. Kurrat Schweinf > A. fistulosum > Arum spp > L. sativum L > A. graveolens > G. tournefortii > M. longifolia > S. oleracea > M. parviflora > B. vulgaris subsp. The reported literature concentrations for Zn are in the range of 9.600–820 µg g−1 [Table 5]. This is comparable to the obtained values of this study. The concentration of Zn in investigated samples is well below the PMTDI of Zn, 0.3–1.0 mg/kg bw.[8] CONCLUSION The present study has generated data on some of heavy metal profile in common indigenous vegetables consumed in Erbil city located in the Kurdistan region of Iraq. The contents were observed and compared with the literature values that have been reported around the world for the same vegetables. Al, Co, Fe, Mn, Ni, Se, and Zn were measured in vegetables using HNO 3 /H 2 O 2 microwave-assisted acid digestion followed by ICP-MS. GBW10015-spinach was used to validate the method. Vegetables of this study showed similar ability to accumulate investigated heavy metals as follows: high quantity for both Al and Fe; medium quantity for Zn and Mn and small quantity for both Co and Se. In addition, Al was found to be at high levels in the majority of vegetables of this study, while Se was below the limit of detection in the majority of investigated vegetables. 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