Analytica Chimica Acta 565 (2006) 81–88

Investigation of four digestion procedures for multi-element determinationof toxic and nutrient elements in legumes by inductively coupled

plasma-optical emission spectrometry

Awad A. Momen, George A. Zachariadis ∗, Aristidis N. Anthemidis, John A. StratisLaboratory of Analytical Chemistry, Department of Chemistry, Aristotle University, Thessaloniki 54124, Greece

Received 15 November 2005; received in revised form 25 January 2006; accepted 31 January 2006Available online 10 March 2006

Abstract

A simple and reliable multi-element procedure for determination of essential (Cr, Cu, Fe, Mg, Mn, Zn) and toxic (Al, Cd, Pb) elements in legumesby inductively coupled plasma-optical emission spectrometry (ICP-OES) was developed. In this contribution, four different digestion procedureswere thoroughly investigated and accurately evaluated with respect to their affect on the analysis of legumes. These included wet digestion withHareatr(o©

K(

1

mlrsinhmhPc

0d

NO3/H2SO4 and HNO3/H2SO4/H2O2, and dry ashing with Mg(NO3)2 and Mg(NO3)2/HNO3. Two calibrations (aqueous standard and standardddition) procedures were studied, and proved that standard addition was preferable for all analytes. ICP-OES operating parameters, such asadio-frequency (RF) incident power, sample uptake flow rate and nebulizer argon gas flow rate were optimized. The precision as repeatability,xpressed as relative standard deviation (R.S.D.) for aqueous standard containing 250 �g l−1 of each analyte was in the range1.5–8.0%. Theccuracy, expressed as relative error was generally varied in the range of 0.5–10% for all analytes, while the quantification limits were lowerhan 2.5 �g g−1. Although, acceptable results were obtained from all developed procedures, wet digestion method with HNO3/H2SO4/H2O2 isecommended for better recovery. The good agreement between measured and certified concentrations with respect to IAEA-331 and IAEA-359CRM’s supplied by IAEA, International Atomic Energy Agency) indicates that the developed analytical method is well suited for determinationf toxic and nutrient elements in legumes and possibly similar matrices.

2006 Elsevier B.V. All rights reserved.

eywords: Sample digestion; Multi-element determination; Toxic and nutrient elements; Legumes; Inductively coupled plasma-optical emission spectrometryICP-OES)

. Introduction

In recent years there has been increasing implementation ofulti-elemental techniques in the analysis of food items to estab-

ish limits for human exposure from the diet. Moreover, differentesearches have been conducted to develop methods to mea-ure constituents in foods which are of interest because of theirmpact in human health [1–4]. Chromium, copper, iron, mag-esium, manganese and zinc are nutrient elements for humanealth, and they are playing an important role in different humanetabolism [4–9]. Environmental pollution is the main cause of

eavy metal contamination in the food chain, and Al, Cd andb are potentially toxic elements that have aroused considerableoncern [10–12].

∗ Corresponding author. Tel.: +30 2310997707.E-mail address: zacharia@chem.auth.gr (G.A. Zachariadis).

Depending on analysis task, many factors should be consid-ered in the choice of sample preparation method such as (a)levels of contamination introduced during sample preparation,(b) degree of volatilization and/or retention during digestionprocess, (c) possibilities to handle a representative sample, (d)completeness of digestion and reproducibility in analyte recov-ery from the matrix, (e) suitability of the analytical techniqueemployed, (f) time needed for sample preparation and (h) eco-nomic aspects including labour and reagent consumption, equip-ment cost, etc. [13–15]. The digestion of organic materials infood samples can be achieved by either dry ashing and/or wetdigestion. Although, dry ashing is well established for the diges-tion and determination of toxic and nutrient elements with mostanalytical techniques, it is associated with losses by volatiliza-tion and/or retention problems. However, these problems canbe overcome or reduced by using of oxidizing materials (ash-ing aids) such as Mg(NO3)2, HNO3 and H2SO4 or a mixtureof MgO/HNO3 or sometime, by careful manipulation of ashing

003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2006.01.104

82 A.A. Momen et al. / Analytica Chimica Acta 565 (2006) 81–88

temperature [16,17]. In addition, the lower blank levels and thecapability in handling large sample, up to 10 g, sometimes makedry ashing methods desirable for toxic and nutrient elementsdetermination. On the other hand, wet digestion methods arewidely used for the digestion of food materials, because of theirsimplicity. They are fairly rapid and flexible in term of beingable to change sample weights and digestion conditions, lessprone to either volatilization or retention losses, and inexpen-sive. The main drawbacks are the co-precipitation of sparinglysoluble compounds, the incomplete digestion of organic materialand the formation of insoluble compounds [18,19]. Correspond-ingly, the use of oxidizing acids such as HNO3 and/or H2SO4is important especially for wet digestion of food samples withhigh carbohydrate and/or fat content, to reduce calcinations andto ensure completeness of the digestion. Also, acid mixtures ofHNO3/HClO4 or HNO3/HCl or combination of more than twoacids are usually employed in wet digestions, while the use ofH2O2 may be needed when the sample material is difficult todigest [20,21].

It important that, the analytical procedure which is used forsample digestion should involve minimal sample handling todecrease the levels of contamination during sample preparation.Moreover, the analytical technique should be rapid, sensitive,with low quantification limits and wide dynamic range, etc.[13,16,22]. Considering these requirements, ICP-OES is a con-venient technique for this task (analysis), because it allows amce

bgaaaOMlOdlb

2

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Oa(((IIMZ

Table 1Operating conditions and description of ICP-OES instrument

RF generator 40 MHz (free running)RF incident powera 1300 WTorch alumina injector, i.d. 2.0 mmNebulizer argon gas flow ratea 0.85 l min−1

Argon gas flow rates 1.5 l min−1 (auxiliary); 15 l min−1

(plasma)Signal measurement mode Peak area (three points per peak)Spray chamber Scott double-passNebulizer type Gem tips cross-flowPump Peristaltic, three channelSample uptake flow ratea 1.0 ml min−1

Polychromator Echelle gratingResolution 0.006 nm at 200 nmDetector Segmented-array charge-coupled (SCD)

235 sub-arrays

a Optimized value.

Muffle furnace (Stuart Scientific Co. Ltd., England) has beenused for ashing. Hot plate and drying oven (Thermolyne, SybronCorporation) were also used. A peristaltic pump was used tointroduce the sample solutions into the ICP and to discard thewastes. To avoid contamination before use, all glassware, diges-tion vessels and storage bottles (which are used for samples,reagents and reference solutions preparations and storage) weresoaked in freshly prepared 10% (v/v) HNO3 for at least 48 h,and finally washed three times with double de-ionized water(DDW). Crucibles were immersed in diluted HCl for about 2days, and rinsed with DDW several times to be used for the nextexperiment.

2.2. Reagents and reference solutions

All chemicals were of analytical-reagent grade andwere purchased from Merck (Darmstadt, Germany), unlessstated otherwise. Multi-element working standard solutionswere prepared by appropriate dilution of stock ICP multi-element standard IV (23 elements) containing 1000 mg l−1

for each element in 0.8 M HNO3. Mineral acids, chem-ical reagents, and oxidizing agents [65% (m/m) HNO3(d = 1.40 kg l−1), 37% (m/m) HCl (d = 1.19 kg l−1), 30% (m/m)H2O2 (d = 1.11 kg l−1), 97% (m/m) H2SO4 (d = 1.84 kg l−1),absolute ethanol (d = 0.79 kg l−1), (Mg(NO3)2·6H2O)] were

ulti-element determination in a single solution, with suffi-iently low quantification limits, sensitive, wide dynamic range,tc. [14,17,23].

In the present work, different digestion procedures that haveeen used for food samples digestion were carefully investi-ated and accurately evaluated with respect to their affects on thenalysis of legume samples. The analytical performances, suchs limits of quantification, precision of the overall proceduresnd accuracy were assessed statistically to optimize the ICP-ES parameters and to evaluate the investigated procedures.oreover, the present study is focused on the mineral content in

egumes, analyzed in digested samples with a validated ICP-ES method under optimized conditions. We also evaluatedifferent digestion methods to develop and recommend an ana-ytical procedure for the digestion of legume samples, followedy the determination of selected toxic and nutrient elements.

. Experimental

.1. Instrumentation and apparatus

All experiments were carried out using a Perkin-Elmerptima 3100XL axial viewing ICP-OES, according to oper-

ting conditions given in Table 1. The analytical wavelengthsnm) were set at the following two different spectral atomicI) and ionic (II) lines for each analyte: Al I (308.215), Al I237.313), Cd II (214.440), Cd II (226.502), Cr II (283.563), CrI (284.325), Cu I (324.752), Cu II (224.700), Fe II (238.204), FeI (239.562), Mg II (279.077), Mg II (280.271), Mn II (257.610),

n II (259.372), Pb II (220.353), Pb I (217.000), Zn I (213.857),n II (202.548).

used. Magnesium nitrate ethanolic solution [(5% (m/v)Mg(NO3)2·6H2O) in ethanol] was also used. All solutions wereprepared with highly purity double de-ionized water (DDW),which was prepared by successfully passing tap water throughtwo ion exchange columns (resin). Also, DDW has been used forwashing and rinsing of all apparatus and glassware. IAEA-331(spinach leaves) and IAEA-359 (cabbage) supplied by IAEA(International Atomic Energy Agency, Monaco) were used foroptimizing, setting up and validation of the whole analytical pro-cedures (i.e., the wet digestion and the dry ashing procedures).The certified reference (or information) values are available formost of analytes under investigation for assessment of the meth-ods accuracy.

A.A. Momen et al. / Analytica Chimica Acta 565 (2006) 81–88 83

2.3. Preparation of sample solutions

Five types of legumes (white bean, faba bean, lentil, greenpea and chickpea) were purchased from the market. Sampleswere selected carefully, to represent the major categories thatavailable in the market. The green pea was refrigerated in thelaboratory until sample preparation. Since the grinding of sam-ples help the digestion process, samples were first shelled,when necessary, grounded in a homemade polyethylene mor-tar and pestle and sieved into fine powders. Ten sub-samples(i.e., different independent samples) from each type were driedto constant mass, cooled and weighed as soon as they reachedroom temperature for further sample pre-treatment. The frozengreen pea was first dried, then grounded and sieved using thesame process that applied for other samples. IAEA-331 andIAEA-359 were used as bottled, without further grinding andsieving, but before digestion or ashing, they were dried asdescribed in the certificates of analysis and recommended by thesupplier.

2.3.1. Wet digestion proceduresAbout 0.5 g sample was weighed into dry, clean PTFE

digestion vessel. One milliliter of DDW was added firstand then supplied with appropriate digestion mixture:HNO3/H2SO4, 2 + 1 (v/v) for wet digestion method 1 (WD1),and HNO /H SO /H O , 4 + 1 + 1 (v/v) for WD2. When theiapctmfissatoAwp

taadA

2

maaostt

furnace at 450 ± 20 ◦C for about 2–3 h for sample ashing. Afterashing was completed (white or a semi-gray ash), the cruciblewas left to cool. If carbon particles remained the crucible wasmoistened with a several drops of HNO3 and the suspension wasagain re-ashed for another 30 min to the same temperature. Thisprocedure yielded a white or a semi-gray ash. The ash was firstmoistened with a few drops of DDW, then dissolved by subse-quently adding 2.5 and 1.5 ml of HCl and HNO3 to the crucible,and finally, quantitatively transferred to 50 ml with DDW. Thesame digestion procedures were carried out for blank solution,and CRM’s to ensure the quality of the analytical procedures.DA1 was based on the dry ashing procedure which described byCoco et al. [27] with minor changes, while, DA2 is the dry ash-ing procedure that described by Adeloju et al. [28] with majormodifications.

Matrix effect studies were carried out by spiking some of theoriginal un-digested samples with variable amounts of standardsolution of the metals. The spiked samples were digested usingthe same ashing procedures as were applied to the non-spikedsamples. All digestions were performed in triplicate.

2.4. Statistical analysis

Differences between mean values were evaluated by Stu-dent’s paired t-test. Linear regression statistical test and cor-relation analysis were performed for calculation of slope (b),ilo

3

3

asllraTbmss(IZaawaohse

3 2 4 2 2nitial reaction has subsided, vessel contents were gently mixednd heated, first at low temperature (about 125 ◦C), then the tem-erature was gradually increased until heavy evolution of fumeseases. If carbonization appeared, the vessel was removed fromhe hot plate, cooled and about 1 ml of HNO3 was added. Theixture was then heated again until complete digestion. Thenal residue (sulfuric acid and inorganic constituent) was dis-olved in 2.5 ml of HCl and diluted to 50 ml with DDW. Theame digestion procedures were carried out for blank solution,nd CRM’s (IAEA-331, IAEA-359) to validate the quality ofhe analytical procedures. WD1 was partly modified from thatf Tinggi et al. [24] and the procedure that recommended byOAC [25] while, WD2 was based on that of D’llio et al. [26]hich applied for determination of trace elements in food sam-les, with major modifications.

Matrix effect studies were carried out by spiking some ofhe original un-digested samples (accurately weighed differentmounts) with variable amounts of standard solution of the met-ls. The spiked samples were then mineralized using the sameigestion procedures as were applied to the non-spiked samples.ll digestions were performed in triplicate.

.3.2. Dry ashing proceduresAbout 3 g sample was weighed into a porcelain crucible. Two

illiliters of DDW were added first, and then supplied withppropriate ashing aid: ethanolic solution of Mg(NO3)2 for dryshing method 1 (DA1), and (5%, v/v) HNO3/ethanolic solutionf Mg(NO3)2, 2 + 1 (v/v) for DA2. When the initial reaction hasubsided, the crucible was heated at low temperature (100 ◦C)o evaporate moisture and excess reagents and calcination ofhe sample. Then, the crucible was transferred into the muffle

ntercept (a) and correlation coefficient (r) of the regressionines. Statistical analysis was based in triplicate measurementsf all samples.

. Results and discussion

.1. Selection of spectral emission lines

All analytes were measured in two different emission lines,nd the main criteria to select between them are the possiblepectral interferences and the concentration range of the ana-yte. In any case the sensitivities were calculated at spectralines of no spectral interferences (with high sensitivity). Theesults of regression analysis of aqueous standard and standardddition solutions of lentil sample using WD2 are presented inable 2, including slope (b), the 95% confidence intervals ofand r-values. The slope of the regression line was used as aeasure of the sensitivity for each analyte and the r as a mea-

ure of the correlation between data. Therefore, in the presenttudy the following spectral lines (in nm) were selected: Al I237.313), Cd II (226.502), Cr II (284.325), Cu I (324.752), FeI (238.204), Mg II (279.077), Mn II (259.372), Pb I (217.000),n I (213.857). The dynamic range of all analytes is extendedt least up to 1 mg l−1, and is considered as sufficient for theccurate quantification of analytes in various legume samples,hich commonly contain lower concentrations of the examined

nalytes. In Table 2, the b-values (for the two emission lines)f all analytes that obtained for aqueous solutions were 1–18%igher than those obtained for standard addition solutions, withome exceptional cases for Cd, Cr, Fe, Cu and Mn. These differ-nces may results from spectral and/or chemical interferences.

84 A.A. Momen et al. / Analytica Chimica Acta 565 (2006) 81–88

Table 2Regression data for ICP-OES at two different wavelengths for aqueous standards and standard addition solutions

Element Wavelength (nm) Aqueous standards Standard addition solutions of lentil sample

Slope (cps �g−1 l) 95% confidenceinterval of slope

r Slope (cps �g−1 l) 95% confidenceinterval of slope

r

Al 308.215 (I) 2.89 ±0.27 0.998 2.49 ±1.19 0.988237.313 (I) 3.41 ±0.61 0.998 2.58 ±0.18 0.999

Cd 214.440 (II) 5.38 ±0.51 0.999 4.65 ±0.92 0.998226.502 (II) 6.22 ±0.95 0.999 7.00 ±0.15 0.999

Cr 283.563 (II) 27.9 ±2.4 0.899 36.8 ±3.3 0.999284.325 (II) 46.5 ±4.8 0.954 45.2 ±1.3 0.999

Cu 324.752 (I) 148 ±15 0.997 152 ±3 0.999224.700 (II) 7.03 ±0.86 0.996 6.73 ±0.19 0.999

Fe 238.204 (II) 37.2 ±2.1 0.999 36.2 ±3.4 0.998239.562 (II) 1.38 ±1.07 0.873 2.08 ±0.51 0.985

Mg 279.077 (II) 5.33 ±3.55 0.977 5.24 ±4.58 0.979280.271 (II) 5.03 ±0.37 0.999 4.93 ±4.53 0.975

Mn 257.610 (II) 114 ±2 0.999 109 ±3 0.999259.372 (II) 128 ±5 0.999 132 ±2 0.999

Pb 220.353 (II) 0.60 ±0.09 0.994 0.41 ±0.09 0.987217.000 (I) 0.69 ±0.16 0.982 0.40 ±0.03 0.999

Zn 213.857 (I) 27.6 ±2.0 0.997 23.3 ±0.3 0.999202.548 (II) 12.9 ±0.7 0.999 12.0 ±0.2 0.999

This primary observation indicates that the sensitivities of thetwo techniques were quite different.

3.2. Optimization of ICP-OES parameters

Since signal intensities of each analyte depend on the samplematrix, it is important to optimize ICP-OES parameters such asthe radio-frequency (RF) incident power, sample uptake flowrate, nebulizer argon gas flow rate, auxiliary argon gas flow rate,plasma argon gas flow rate, etc. Emission intensities are mostlyaffected by the first three parameters which have a greater impactupon the sensitivity of the measurement, whereas other param-eters have relatively small effects on sensitivity and are usuallyadjusted to accommodate memory effects due to particular sam-ple type such as organic materials and/or dissolved solids. In thisstudy two calibration procedures (aqueous standard and standardaddition) were studied using lentil digest solution to select thecalibration techniques, while IAEA-359 digest solution was uti-lized to optimize the ICP-OES operating parameters. The RFincident power was optimized as it affects seriously the plasmatemperature and improves atom excitation performance. It wasstudied in the range 1100–1500 W. The results indicate that thesensitivity and the linearity are better at 1300 W for almost allanalytes, and the stability of plasma was also improved (Fig. 1).Moreover, the sample uptake flow rate was investigated at threelevels: 1.0, 2.0 and 3.0 ml min−1. It was found that the emis-srsh

flow rate of 1 ml min−1, which provide sufficient sensitivity andlow sample consumptions, was chosen as optimal and appliedfor quantification throughout this study. The effect of nebulizerargon gas flow rate was studied from 0.6 to 1.5 l min−1 appear-ing a maximum intensity at 0.85 l min−1 for all analytes (Fig. 2).Thus, a 0.85 l min−1 nebulizer argon gas flow rate was adoptedthroughout this study, which provides high sensitivity and goodprecision. The optimum values for all parameters that studiedare presented in Table 1.

3.3. Limits of quantification

The solution limits of quantification (LOQ) of the ICP-OESinstrument are calculated as the concentration equal to 10 times

Fia

ion intensities for Cd and Pb were higher at 1 ml min−1 flowate, while for Al, Cr, Cu, Fe, Mg and Mn the flow rates werelightly higher at 2 ml min−1 and only for Zn the intensity wasigher at 3 ml min−1. Therefore, in the present study, sample

ig. 1. Effect of RF incident power on the emission intensity of Mg, Cu, Zn, Crn IAEA-359 digest solution using HNO3/H2SO4/H2O2. All other parameterss in Table 1.

A.A. Momen et al. / Analytica Chimica Acta 565 (2006) 81–88 85

Fig. 2. Effect of nebulizer argon gas flow rate on the emission intensity of Mg,Cu, Fe, Mn in IAEA-359 digest solution using HNO3/H2SO4/H2O2. All otherparameters as in Table 1.

the standard deviation of 10 blank measurements divided by theslope of calibration line of each analyte. It represents the low-est concentrations of analytes in legume samples (�g g−1) thatcan be quantified with the referred instrument. For the calcula-tion of the quantification limits, 10 separates blank solutionswhich prepared independently were analyzed. The obtainedquantification limits (Table 3) are generally provide sufficientquantification capability to determine almost all analytes (withsome exceptional cases for Cd, Cr and Pb) at the concentrationspresented in analyzed samples with ICP-OES technique. Fur-ther improvement of quantification limits values for the solidmaterial may be achieved by using larger sample weights and/orlower final dilutions. So, by this way the concentration of Cd andCr can be determined (quantified) in some legume samples usingdry ashing methods. On the other hand, for the selected emis-sion lines of all analytes the b-values that obtained for aqueoussolutions were 1–15% higher than those obtained for standardaddition solutions, with some exceptional cases for Cd, Cu andMn. These differences may results from spectral or chemicalinterferences. This primary observation indicates that the sen-

sitivities of the two techniques were quite different. Thus, theuses of standard addition techniques were important to elimi-nate these interferences. The calculated R.S.D. for blank and foraqueous standard containing 250 �g l−1 of each analyte werefound in the range between 1.3 and 7.5% and 2.0–8.1%, respec-tively (n = 10). The results indicate that the procedures offergood repeatability (precision) for all analytes (R.S.D. lower than8.5%).

3.4. Efficiency of digestion procedures and accuracycontrol

To verify the accuracy of the proposed procedures, twoCRM’s: spinach leaves and cabbage have been treated by theproposed procedures and analyzed by ICP-OES using the oper-ation conditions that listed in Table 1. These reference materialswere chosen as they were the closest available to food samples(all of them are of plant origin). Also, they are certified for mostof the analytes of interest to be determined. The results of anal-ysis indicated that there is a good agreement between measuredand certified values within a relative errors of less than 16%, witha few notable exceptions for Cd and Cu (Tables 4 and 5). More-over, Student’s t-test at 95% confidence level showed that therewas insignificant difference between the means of the certifiedand obtained values for almost all analytes under investigation.According to the recovery values, and the relative error per-comteepapFt

Table 3Slopes and limits of quantification (LOQ, calculated as �g l−1 and expressed as �g g

Element, spectral line (nm) Slope (cps �g−1 1) Limits of qua

s stan)

ACCCFMMPZ

lanklanklank

Aqueousstandards

Standardadditiona

Aqueou(�g l−1

l I (237.313) 3.41 2.58 6.7d II (226.502) 6.22 7.0 4.0r II (284.325) 46.5 45.2 6.2u I (324.752) 148 152 2.7e II (238.204) 37.2 36.2 8.2g II (279.077) 5.33 5.24 9.0n II (259.372) 128 132 0.5

b I (217.000) 0.69 0.41 24.5n I (213.857) 27.6 23.3 9.5

a Lentil sample was used for the standard addition technique.b Concentration corresponding to 10 standard deviation of the signals of the bc Concentration corresponding to 10 standard deviation of the signals of the bd Concentration corresponding to 10 standard deviation of the signals of the be Not quantitated due to Mg(NO3)2 addition as ashing aid.

entages with respect to CRM’s, no significant differences werebserved in efficiency between wet digestion and dry ashingethods as indicated by Student’s t-test. Also it was observed

hat wet digestion procedures gave the best results (higher recov-ries) for the majority of the analytes. Although sufficient recov-ries were obtained for almost all analytes using the proposedrocedures, it was found that WD2 procedure give the mostccurate and reproducible analytical data comparing to otherrocedures (relative error of less than 11%) (Tables 4 and 5;ig. 3). This indicates that, the presence of H2O2 is very impor-

ant for digesting food samples high in carbohydrates or fats.

−1) for wet digestion and dry ashing methods

ntification

dardsb Equivalent to wet digestionmethodc (�g g−1)

Equivalent to the dryashing methodd (�g g−1)

0.67 0.110.4 0.070.62 0.100.27 0.050.82 0.140.9 NQe

0.05 0.012.5 0.410.95 0.16

solutions.related to a sample mass of 0.5 g finally diluted in 50 ml.related to a sample mass of 3.0 g finally diluted in 50 ml.

86 A.A. Momen et al. / Analytica Chimica Acta 565 (2006) 81–88

Table 4Accuracy of the analytical methods for determination of analytes in IAEA-331 using various digestion techniquesa

Element Certified valueb (�g g−1) Foundc (�g g−1) (recovery, %)c

WD1 WD2 DA1 DA2

Al 310.0 ± 12.0 250.0 ± 7.2 (80.7 ± 2.3) 276.0 ± 8.1 (89.0 ± 2.6) 255.0 ± 8.2 (82.3 ± 2.6) 270.0 ± 10.9 (87.1 ± 3.5)Cd 2.89 ± 0.06 2.73 ± 0.13 (94.5 ± 4.6) 2.98 ± 0.16 (103 ± 6) 2.20 ± 0.15 (76.1 ± 5.1) 2.25 ± 0.14 (77.9 ± 4.9)Cr NAd 2.57 ± 0.17 2.45 ± 0.15 2.60 ± 0.16 2.32 ± 0.15Cu 12.20 ± 0.60 12.10 ± 0.65 (99.2 ± 5.3) 12.0 ± 0.6 (98.4 ± 5.2) 14.52 ± 0.66 (119 ± 5) 14.97 ± 0.69 (123 ± 6)Fe NA 266.0 ± 7.9 273.0 ± 8.6 283.3 ± 8.4 293.0 ± 9.8Mg 8900 ± 0.01 8850 ± 227 (99.4 ± 2.6) 915 ± 244 (103 ± 3) NQe NQMn 75.90 ± 2.30 76.35 ± 3.40 (101 ± 5) 76.10 ± 2.87 (100 ± 4) 81.0 ± 3.8 (107 ± 5) 82.50 ± 3.95 (109 ± 5)Pb NA <LOQf <LOQ <LOQ <LOQZn 82.0 ± 3.30 76.20 ± 3.72 (92.9 ± 4.5) 78.20 ± 3.96 (95.4 ± 4.8) 83.83 ± 4.18 (102 ± 5) 85.17 ± 4.24 (104 ± 5)

a WD1, WD2 (wet digestion); DA1, DA2 (dry ashing).b IAEA-331 certified values (mean ± standard deviation).c Mean ± standard deviation (n = 3).d Not available.e Not quantitated due to Mg(NO3)2 addition as ashing aid.f Results were lower than the limits of quantification of ICP-OES.

Table 5Accuracy of the analytical methods for determination of analytes in IAEA-359 using various digestion techniquesa

Element Certified [or information]valueb (�g g−1)

Foundc (�g g−1) (recovery, %)c

WD1 WD2 DA1 DA2

Al NAd 108.36 ± 4.47 105.70 ± 4.42 107.53 ± 4.51 98.33 ± 3.95Cd 0.120 ± 0.005 <LOQe <LOQ 0.09 ± 0.01 (75.1 ± 3.3) 0.10 ± 0.01 (81.4 ± 3.3)Cr [1.30 ± 0.06] 1.32 ± 0.07 (100 ± 5) 1.41 ± 0.07 (109 ± 6) 1.29 ± 0.07 (99.2 ± 5.0) 1.23 ± 0.06 (94.6 ± 4.8)Cu [5.670 ± 0.180] 5.59 ± 0.20 (98.6 ± 3.6) 5.65 ± 0.25 (99.6 ± 4.4) 6.70 ± 0.23 (118 ± 4) 6.80 ± 0.28 (120 ± 5)Fe 148.0 ± 3.9 127.0 ± 5.4 (85.8 ± 3.6) 149.0 ± 4.9 (101 ± 3) 154.2 ± 6.0 (104 ± 4) 148.0 ± 5.8 (100 ± 4)Mg 2160 ± 50.0 1890.0 ± 47.3 (87.5 ± 2.2) 2070.0 ± 53.1 (95.8 ± 2.5) NQf NQMn 31.90 ± 0.60 28.80 ± 1.48 (90.3 ± 4.6) 31.30 ± 1.62 (98.1 ± 5.1) 35.33 ± 1.67 (111 ± 5) 32.83 ± 1.64 (103 ± 5)Pb NA <LOQ <LOQ <LOQ <LOQZn 38.60 ± 0.70 39.40 ± 2.11 (103 ± 6) 38.50 ± 2.04 (99.7 ± 5.3) 42.83 ± 2.12 (111 ± 6) 40.83 ± 2.11 (106 ± 6)

a WD1, WD2 (wet digestion); DA1, DA2 (dry ashing).b IAEA-359 certified (or information) values (mean ± standard deviation).c Mean ± standard deviation (n = 3).d Not available.e Results were lower than the limits of quantification of ICP-OES.f Not quantitated due to Mg(NO3)2 addition as ashing aid.

Fig. 3. Comparison of relative error (%) encountered in the determination of several elements from IAEA-331 (left) and IAEA-359 (right) CRM’s usingHNO3/H2SO4/H2O2 as digestion mixture.

A.A. Momen et al. / Analytica Chimica Acta 565 (2006) 81–88 87

This is in agreement with the results of D’llio et al. [26] whichapplied a similar procedure for the determination of trace ele-ments in food samples.

3.5. Study of matrix effect in legume samples

To determine if the sample matrix could interfere with theanalytes causing spectral overlaps, linear regression statisticaltest at 99% probability level was applied. Slope (b) and inter-cept (a) of comparisons of the regression lines were calculatedby plotting the emission intensities of aqueous standards versusthe emission intensities of standard addition solutions (Table 6).The calculated b-values were in the range of 0.8–1.2 cps �g−1 l(counts per second �g−1 l) for all analytes with some excep-tional cases. The theoretical model assumes that, the value of bis equal to unity (absence of systematic or random errors) andthe value of a is equal to zero (absence of bias) [29]. Practi-cally, if the observed b-value did not differ significantly fromunity then the sensitivities between the two techniques weresimilar, otherwise the two techniques were significantly differ-ent. It was found that the analytes were shown significant (S)or non-significant (NS) differences (at 99% probability level)depending on type of sample matrix and the digestion proce-dure that applied. These differences may attribute to both thedigestion mixtures and sample matrices, which causes chemi-cal and spectral interferences, respectively. Moreover, the the-o(rlrudomtfa

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retical model assumes that, the value of r is equal to unitymaximum accordance) [29]. It was found that the calculated-values varied in the range of 0.813 and 0.999 for all ana-ytes. Also, these differences may be attributed to the sameeasons that are mentioned above. Consequently, calibrationssing the standard addition technique are recommended foretermination of these elements in legume samples, to overcomer to eliminate most of the errors due to non-linearity and/oratrix interferences that causes low sensitivity. On the overall,

he results proved that, all procedures are in good accordanceor analyzing legume samples with acceptable precision andccuracy.

.6. Applications

The recommended analytical method using HNO3/H2SO4/2O2 as digestion mixture was applied for the determination ofl, Cd, Cu, Cr, Fe, Mg, Mn, Pb and Zn in five types of the most

ommonly consumed commercial legume samples (Table 7).he results showed that the concentration of Cd in both greenea and chickpea was lower than the limits of quantification ofCP-OES, while, the concentrations of both Cr and Pb in allegume samples were lower than the limits of quantification.lternatively, such Cd and Cr concentrations can be determinedsing the dry ashing methods in legumes and possibly similaratrices. Moreover, it can be summarized that the concentration

f toxic (Cd, Al) and nutrient (Fe, Cu, Mg) elements in the exam-ned samples is approximately similar when comparing them tohe concentration of these elements in legume samples availablen various part of the world, as it was previously reported initerature [30].

88 A.A. Momen et al. / Analytica Chimica Acta 565 (2006) 81–88

Table 7Analytes concentrations in legumes using HNO3/H2O2/H2SO4 digestion mixture

Element Concentrationa (�g g−1)

White bean Faba bean Lentil Green pea Chickpea

Al 13.0 ± 0.5 17.0 ± 0.5 39 ± 3 1.0 ± 0.1 35 ± 3Cd 0.90 ± 0.08 0.80 ± 0.06 0.50 ± 0.03 <LOQ <LOQCr <LOQb <LOQ <LOQ <LOQ <LOQCu 15.7 ± 1.0 16.2 ± 1.0 7.7 ± 0.4 20 ± 1 13.0 ± 0.6Fe 79 ± 4 110 ± 5 129 ± 5 23 ± 2 64 ± 3Mg 1524 ± 35 1361 ± 30 2229 ± 59 1688 ± 44 1596 ± 39Mn 23 ± 2 15.0 ± 0.5 19.0 ± 0.8 9.0 ± 0.4 33 ± 3Pb <LOQ <LOQ <LOQ <LOQ <LOQZn 82 ± 4 34 ± 3 58 ± 3 42 ± 3 45 ± 2

a Mean ± standard deviation (n = 3).b Results were lower than the limits of quantification of ICP-OES.

4. Conclusions

The analytical ICP-OES method that has been developedand validated for legume samples proved to be simple (easyprocedure: 0.5 g sample + HNO3/H2SO4/H2O2, 4 + 1 + 1, v/v),accurate (relative error less than 11%), precise (R.S.D.% lowerthan 8.5%) and reliable (highly confidence procedure) for simul-taneous determination of toxic and nutrient elements in legumesamples. Also, they can be considered as useful techniques forroutine analysis of toxic and nutrient elements in foods and pos-sibly similar matrices and in quality control. Calibration afterstandard addition was preferable to those using aqueous stan-dards. The dynamic range of the methods covers the range ofthe concentrations usually found in legume samples. Moreover,although, acceptable results were obtained from all developedprocedures, wet digestion method with HNO3/H2SO4/H2O2 isrecommended for legumes to obtain better analyte recovery. Theconcentration of toxic and nutrient elements in our samples isapproximately similar to the level in legumes from surroundingAsian and European countries.

Acknowledgment

The financial support of this research by Greek State Schol-arship Foundation (IKY) is gratefully acknowledged.

R

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