Determination of 25 trace element concentrations in biological reference materials by ICP-MS following different microwave-assisted acid digestion methods based on scaling masses of digested samples

Hindawi Publishing Corporation ISRN Analytical Chemistry Volume 2013, Article ID 851713, 14 pages http://dx.doi.org/10.1155/2013/851713 Research Article Determination of 25 Trace Element Concentrations in Biological Reference Materials by ICP-MS following Different Microwave-Assisted Acid Digestion Methods Based on Scaling Masses of Digested Samples S. M. Enamorado-Báez,1,2 J. M. Abril,2and J. M. Gómez-Guzmán3 1Centro Nacional de Aceleradores (CNA), Universidad de Sevilla, Thomas Alba Edison 7, 41092 Seville, Spain 2Departamento de Fisica Aplicada I, E.T.S. de Ingenieria Agronomica, Universidad de Sevilla, Carretera Utrera km. 1, 41013 Seville, Spain 3Technische Universit ¨ at M¨ unchen, Physics Department E12, James-Franck-Straße 1, 85748 Garching bei M¨ unchen, Germany Correspondence should be addressed to J. M. G´ omez-Guzm´ an; [email protected] Received 28 April 2013; Accepted 26 May 2013 Academic Editors: H. Alemu, G. Drochioiu, D. J. Fletouris, F. Kandemirli, A. Orte, and I. Zhukov Copyright © 2013 S. M. Enamorado-B´ aez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The use of normalized procedures designed for soil and sediment samples (like US-EPA 3051) to chemically prepare some kind of organic samples is a common practice in some laboratories. However, the performance of this method for other matrices has to be demonstrated. Three microwave-assisted digestion procedures with 0.5 g of sample and simplified reagents (10 mL HNO3alone and mixtures of HNO3/HCland HNO3/H2O2procedures A, B, and C, resp.) were compared for quantitative determination of 25elements(Be,B,Al,Ti,V,Cr,Mn,Fe,Co,Ni,Cu,Zn,As,Se,Sr,Mo,Ag,Cd,Sb,Cs,Ba,Tl,Pb,ThandU)inthreebiological reference materials provided by NIST (mussel tissue (MT), tomato leaves (TL), and milk powder (MP)) by ICP-MS. From scaling masses (from 0.1 up to 0.9 g at 0.1 g interval) in procedure A, a linear relationship among instrumental signal and mass of digested sample could be constructed at 99% CL for most of the target analytes. The slope of this linear fit provided the estimation of sample concentration, while the ordinate in origin allowed the identification of matrix interferences which were absent in the reagent blank. 1. Introduction Inductively coupled plasma mass spectrometry (ICP-MS) is a robust and widely used technique for multielemental and isotopic analysis of environmental materials [1–3]thathas shown clear advantages when compared with other analytical techniques such as inductively coupled plasma atomic emission spectrometry (ICP-AES) [4–6], flame atomic absorption spectrometry (F-AAS), and electrothermal atomic absorption spectrometry (ET-AAS) [7,8]. The basic setup for ICP-MS analysis requires the sample introduction as a liquid solution and thus, for solid matrices, an acid digestion procedure becomes mandatory. Sample digestion is mainly carried out by a fusion or a wet procedure based on an acid digestion with a heated mixture of mineral acids [2,9–13]. In general, closed digestion systems are to be preferred to minimize possible contamination of the digest, increase reproducibility, and avoid losses of volatile elements [14–17]. Wet microwave digestion equipped with temperature and pressure control assisted by common mineral acids, such as nitric, sulphuric, perchloric and hydrochloricacids,isfrequentlyusedforsampledigestion[18]. In order to dissolve the silicates and eliminate the effects of silica gel in environmental samples, hydrofluoric and orthoboric acids are usually used, although they can produce unsatisfactory recoveries in volatile elements [19,20]. A mixture of nitric acid and hydrogen peroxide is widely employed because they mineralise organic matter effectively and produce less spectral interference in ICP analyses [1]. Nitric acid has been reported to be strong enough to solubilize metals 2ISRN Analytical Chemistry from fly ashes [21], from soils with organic carbon content up to 38% [22], and from plant materials for environmental monitoring [23]. However, in general, plant samples require a more complete decomposition procedure due to the presence of high organic and/or silicon contents [24]. The use of normalized procedures for soil and sediment samples to prepare some kind of organic sample is a common practice. The US-EPA 3051 [25] proposes to use a representative amount of sample of 0.5 g digested with 10 mL of concentrated nitric acid. For some cases this method also proposes theuseofthesameamountofsampledigestedinamixture of 9 mL of concentrated nitric acid and 3 mL of concentrated hydrochloric acid. In the US-EPA 3052 [26]method,arepresentativesampleofupto0.5gisdigestedin9mLofconcentrated nitric acid, and usually 3 mL hydrofluoric acid, although the method has provisions for scaling the sample sizeuptoamaximumof1.0gandenablestheanalystto select other decomposition reagents. The use of hydrofluoric acid requires strict safety procedures, and it can damage the glass components during instrumental analysis if its excess was not previously removed from the samples. Apart from the normalized US-EPA methods, other digestion methods can be extracted from the specialized literature and used with particular samples [27]. Thus, in this work, we will study the performance of methods based on the use of just nitric acid compared with those using a combination of nitric acid and hydrochloric acid or hydrogen peroxide. These options were chosen on the basis of widespread usage and seemingly minimal contribution to ICP-MS spectral interferences. In this work, we will be concerned with the analysis of biological samples with different characteristics to show the versatility of the procedures, although this is far away from being a universal procedure due to the limited number of certifiedmaterialsused.Theaciddigestionmethodsproposedin the literature are used in the preparation of soil, sediments, and other complex matrices for a limited number of elements. The US-EPA methods pursuit the acid digestion and allow the quantificationofupto26elements(Al,Sb,As,B,Ba,Be,Cd, Ca,Cr,Co,Cu,Fe,Pb,Mg,Mn,Hg,Mo,Ni,K,Se,Ag,Na,Sr, Th,V,andZn).Theperformanceofthismethodforotheranalytes and/or matrices has to be demonstrated. Instrumental developments in both, ICP-MS and microwave digestion systems, and the need of limiting the use of hazardous acids lead to an increasing interest in the improvement and updating of sample digestion methods [1,2,28–32]. Until now, standard methods for soils and/or sediments have been directly applied to the preparation of organic samples. In this work, we will try to justify the use of those methods for organic samples and, depending on the results obtained, determine another alternative method which could give better results. For that reason, we have made a digestion procedure using only nitric acid (similar to the US-EPA 3051 method established for sediments, sludges, soils, and oils) but increasing the sample mass to acid volume ratio. Also, digestion procedures based on the use of a combination of nitric acid and hydrochloric acid or hydrogen peroxide have been checked. The effect of microwave digestions with different reagents will be tested on 25 different elements determined through the ICP-MS technique in three different biological standard reference materials (SRMs) provided by the National Institute of Standards and Technology (NIST). The amount of sample willbescaledfrom0.1upto0.9gusingjustnitricacid.The study of the instrumental signal versus the sample mass will enable a quality test, when a good linearity is found, and will allow the determination of analyte concentration in the sample from the slope. Furthermore, an ordinate in origin different from zero at a fixed confidence level will serve to identify matrix effects noncompensated by a background subtraction based upon conventional digestion (reagents) blanks. Comparison between direct determination of concentrations based on dilution factors and the corresponding determined from the linear fit will serve to identify the mass distribution with unacceptable results. This, along with the study of relative uncertainties, will allow the construction of a figure of merit to find out the most suitable sample amount in the digest. Finally, a comparative study of three different digestion procedures applied to the SRM samples will be accomplished. 2. Experimental 2.1. Sample Materials and Reagents. In this work, the following biological standard reference materials (SRMs) provided by the National Institute of Standards and Technology (NIST) were used: SRM 1549 nonfat milk powder (referred hereafter as MP) [33], SRM 2976 mussel tissue (referred hereafter as MT) [34],andSRM1573atomatoleaves(referredhereafter as TL) [35]. SRM 1549 was prepared by NIST to provide assistance in overcoming the difficulties in accurate trace and ultratrace levels analyses of food and other biological important materials (some of its certified elements have concentrations below 0.01 mg kg−1), and its certified major constituents with concentrations above 1% (dry mass basis) are calcium, chlorine, phosphorus, and potassium. SRM 2976 were collected by the International Atomic Energy Agency (IAEA) from the Mediterranean coast of France as part of an effort to investigate metal speciation in the marine environment and its major constituents with concentrations above 1% (dry mass basis) are chlorine and sodium. Finally, SRM 1573a were obtained by the NIST from plants at the Horticultural Research Farm at Rock Springs, PA (USA). It was produced to evaluate the analyses of some elements in botanical materials and agricultural food products, and its certified major constituents with concentrations above 1% (dry mass basis) are calcium, nitrogen and potassium. Allreagentsusedforthemicrowave-assisteddigestions, that is, hydrochloric acid (36% HCl), nitric acid (69% HNO3) and hydrogen peroxide (30% H2O2), were of suprapur grade (Merck, Darmstadt,Germany).High-purity water(18MΩcm) from a Milli-Q water purification system (Millipore, Bedford, USA) was used for dilution of the standards, for preparing samples throughout the chemical process, and for final rinsing of the acid-cleaned vessels, glasses, and plastic utensils. Before use, all glass and plastic utensils were thoroughly acid cleaned and then rinsed with Milli-Q water. Moreover, prior to the use of the tetrafluoroethylene (PTFE-TFM) vessels, the following cleaning procedure was carried out: 10 mL of concentrated HNO3was added to each vessel, and, once ISRN Analytical Chemistry 3 Table 1: Instrumental settings and calibration for ICP-MS. ICP-MS instrument Forward power 1300 W Sampler and skimmer cones Nickel Argon flow rates Cool gas 14.5 L min−1 Auxiliary 0.76 L min−1 Nebuliser 0.92 L min−1 Acquisition parameters Ion monitoring mode Number of sweeps 60 Channels per mass 1 Dwell time 10 ms Number of main runs 3 Survey mode Cannels per mass 10 Sweeps 10 Dwell time 0.6 ms Internal standards 6Li, 45Sc, 115In, 159Tb, 209Bi closed, the temperature was raised to 180∘Cwithin15min andheldatthistemperaturefor10min.Aftercooling,the content of the vessels were discarded; PTFE-TFM vessels were soaked overnight with diluted HNO3andthenwererinsed with double deionised water. The external calibration solutions must include known concentrations of each target analyte. They were prepared from standard certified elemental solutions (Cromlab) and Milli-Q water containing 1% HNO3to get a range of concentrations: 0.5, 2.5, 5.0, 25.0, 50.0, and 250 𝜇gL −1 (for all elements except for Se, which were fivefold higher). A blank solution consisting in Milli-Q water containing 1% HNO3 completed the calibration curve (counts versus 𝜇gL −1)for each analyte. The nonspectral matrix effects associated to the ICP-MS measurements were resolved by the addition of internal standards. The standard solution was prepared by diluting singleelemental stock solutions with Milli-Q water containing 1% HNO3up to get 50 𝜇gL −1 of indium, terbium and bismuth, 500 𝜇gL −1 scandium and 1000 𝜇gL −1 lithium. 2.2. Analytical Instrumentation. An inductively coupled plasma mass spectrometry system Thermo Elemental ICP-MS X7 (Thermo Fisher, Cambridge, UK) with quadrupole mass analyzer, multichannel detector (Pulse Counting and Analog Methods), auto sampler ASX-500 (CETAC, Omaha, NE, USA), and software Plasma Lab version v4.5 was used for this work. The instrument, located at the Servicio de Investigaci´ on Agraria laboratory (University of Seville, Spain), was used withaconcentricMeinhardtypeglassnebulizer,asilicaimpactbead spray chamber,cooledto3∘CbyaPeltiercooler,and a standard silica torch. Standard nickel sample and skimmer cones were used. The internal standard solution was added onlinebya“Y”connectioninthepipewherethesampleis aspired by the peristaltic pump. Table 2: Equations used for the correction of isobaric and polyatomic interferences#. Element 𝑚/𝑧 Correction equation V 51 (1) 51V: −0.35252Cr −3.12753Cr As 75 (2) 75As: −0.03182Kr −3.132277ArCl Se 82 (3) 82Se: −1.00183Kr Cd 111 (4) 111Cd: −0.764106Cd −1.073108Cd Pb 208 (5) 208Pb: 1.00206Pb + 1.00207Pb #With the recommended coefficients for the X-series ICP-MS instruments. The ion optics were tuned to optimise the sensitivity of the signal at 𝑚/𝑧9,59,115,137,140,and238fora100mgL −1 beryllium, cobalt, indium, barium, cerium, and uranium solution, respectively, which was typically 10000–60000 counts s−1 in standard mode. The relative standard deviation of isotopes signals was less than 5%. The oxide and double charged levels were both monitored to ensure that the 140Ce+/140Ce16O+and 137Ba+/137Ba++ ratios did not exceed 2% and 5%, respectively. Instrumental performance optimization, including nebulizer gasflowrate,RFpower,andionlensvoltages,wasperformed and operational conditions are described in Table 1. The US-EPA 200.8 method [36]istheroutineanalytical method implemented in our lab. In its original version, the method is applied for 21 elements, but in this work, we includedB,Ti,Fe,Sr,andCsandexcludedmercury,since this last element requires a separate procedure due to its important memory effects. Thus, this was the list of 25 target analytes:Be,B,Al,Ti,V,Cr,Mn,Fe,Co,Ni,Cu,Zn,As,Se, Sr,Mo,Ag,Cd,Sb,Cs,Ba,Tl,Pb,Th,andU. Although there are recommended targets 𝑚/𝑧in the USEPA 200.8 method, usually several isotopes are monitored for each element. This provides helpful information for the analyst to properly interpret and quantify the acquired data. Thus, 48 𝑚/𝑧 havebeenmonitoredinthemainrunmode, and spectra were acquired in survey mode for each measured sample. These spectra will serve to check interference effects. Finally, the undesirable effects of isobaric and polyatomic interferences were corrected using the interference correction equations given in Table 2. For closed-vessel digestions, a microwave system Multiwave 3000 (Anton Paar, Graz, Austria) with rotor HF100 and software version v1.52 was used. The system was equipped with 16 high-pressure PTFE-TFM vessels with an internal volume of 100 mL (maximum pressure and temperature of 40 bars and 240∘C, resp.). 2.3. Microwave-Assisted Digestion Procedures. Three digestion procedures using different reagents were tested for digestion of standard reference biological materials: procedure A, assisted by HNO3; procedure B, assisted by HNO3and HCl; and procedure C, assisted by HNO3and H2O2.For the previously mentioned procedures, approximately 0.5 g of sample were weighted directly into the PTFE-TFM vessels, to which the reagents were added (10 mL HNO3for procedure A, 10 mL HNO3+3mLHClforprocedureB,and 10 mL HNO3+3mLH 2O2for procedure C) and the vessels 4ISRN Analytical Chemistry were closed immediately. The operational conditions and the heating program used were carried out according to these conditions: a ramp time of 25 min to reach 200∘Canda hold time of 25 min at 200∘C. After cooling the vessels to room temperature, they were vented and opened. In that moment, Milli-Q water was added to the vessels and they were closed and shaken thoroughly to dilute any possible rest of colloids attached in the vessels’ walls. This process was repeated three times. The resultant mixture was filtered with a20–25𝜇mdiameterporefilter110mmdiameter(Whatman) anddilutedto100mLinavolumetricflaskwithMilli-Q water. To accomplish the TDS requirements for sample introduction in our ICP-MS, a further dilution of 1.2 mL (1.0 mL for procedure C) of the previous digested solution to 10 mL of Milli-Q water with 1% suprapure HNO3was needed. Triplicate samples of the three reference materials were prepared by each digestion method. The digestion procedure A was then modified, keeping constant the acid volume (10 mL HNO3)andscalingthe samplemassfrom0.1upto0.9gat0.1gintervals.Triplicate samples of the three reference materials were prepared by this digestion method using the same microwave conditions previously described, and being then subject to the same dilution factor. Matrix spike samples were prepared in duplicate following the US-EPA 200.8 procedure for the three biological matrices for a final extra concentration of 5 𝜇gL −1 for all the analytes (25 𝜇gL −1 for Se). Triplicate digested reagent blank solutions for each digestion procedure were analyzed for determination of the method detection limit (MDL) [2,37]. These reagent blank solutions were ascribed for background correction in the postexperiment analysis. 2.4. Statistical Analysis. Each individual sample was measured with three main acquisition runs during the experiment, providing mean values and standard deviation. Results reported in this work will refer to the mean value and standard deviation of the three replicates of each organic matrix and digestion method. The general linear model procedure in Statgraphics Plus 5.1 (StatPoint 2000) was used. This software was also used for regressions analyses. 3. Results and Discussion 3.1. Recovery of Internal Standard and Quality Controls. Recovery of internal standards decreased monotonically throughout the experiment up to ∼70% of their initial values for 115In, 159Tb, and 209Bi and up to 76% for 45Sc, being this an usual behaviour. The recovery of 6Li increased throughout the experiment to reach approximately a 140% of its initial value (in TL sample). This isotope can be naturally present in thesamples,anditcanbeinterferedby12C2+. Accordingly, 6Li was removed as internal standard in the postexperiment analysis. All target analytes were ascribed to the interpolation mode except Al, Be, B, and Ti, which were directly ascribed to 45Sc. The calibration curve for all the isotopes showed a good linearity over the whole range of concentrations, with correlation coefficients higher than 0.999 except for Fe and Al, which showed some deviations for low concentrations. Results for Alarereported,buttheyhavetobehandledcarefullysince some of the measured concentrations (∼550 𝜇gL −1) were beyond the range of the calibration curve (0.5–250 𝜇gL −1). Quality control (QC) samples included external calibration verification (ICV), initial and continuous blank verification, and matrix spike samples (MXP). All target analytes passed all QC, but the 𝑚/𝑧 = 66 (Zn) failed the ICV test for 5 𝜇gL −1, although it showed a good behaviour for higher concentrations, in the range of the certified target values. 3.2. Reagent Blanks and Method Detection Limit (MDL). Detection limits for each digestion procedure (MDL) were determined from reagent blanks by using the US-EPA 200.8 definitions. Each reagent blank was prepared by using the same volume and acid combinations, and following the same experimental procedure used to prepare the real samples. Results are shown in Table 3. They are reported for the three digestion procedures which used 0.5 g of sample and referred to concentration in original sample (mg kg−1). For the variations made in procedure A with different sample amount, 𝑚𝑖(g), the corresponding MDLs can be obtained by correcting those from procedure A by a factor 0.5/𝑚𝑖.The use of correction equations may affect the MDL, as shown in Table 3. The MDLs reported are about one order of magnitude higher than those reported by Sucharov´ aandSuchara[1] because the dilution factor was also one order of magnitude higher in our case. For most of the elements, the choice of thedigestionprocedurehadlittleinfluenceontheMDL,in agreement with previous results [2]. When reagent blanks were treated as unknown samples with subtraction of the calibration blank, only Al (2.4– 5.5 𝜇gL −1)andCd(∼0.03 𝜇gL −1) could be quantified over the instrument detection limits. The first one is an airborne pollutant,andthesecondoneislikelylinkedtosomecrosscontamination coming from previous tracing studies carried out in our lab. 3.3. Procedure A with Scaling Masses: Linear Regression Method. Concentrations in the digests were determined for each 𝑚/𝑧 ratio in each target analyte by using the corresponding calibration curve from calibration standards and the measured signal (after subtracting the corresponding reagent blank). Then, a mean value and a standard deviation of mean value were obtained from the three analytical replicates. Results were then plotted against the respective digested masses. Figures 1(a) and 1(b) show examples for a selected group of elements measured in MT. Data followed a linear relationship. Linear regression analysis provided the slope and ordinate in the origin (with their corresponding uncertainties) and the correlation coefficient. Concentrations below the detection limit in the digest were excluded from the analysis, applying the regression fit to the remaining data point. Only in few cases the linear relationship did not hold (a confidence level below 90% was used as criteria for rejection), ISRN Analytical Chemistry 5 and the corresponding elements were discarded for quantification (see Tables 4(a), 4(b), and 4(c)). This usually happened for those analytes whose concentrations were below the detection limit for all or most of the masses (e.g., Ag or Cs in MT). The element 47Ti was interfered by 40Ar + 7Li, being this last added as internal standard. Formostofthe𝑚/𝑧 ratios, the linear relationship held at 99% CL (Table 4), with correlation coefficients over 0.97. Results are summarised in Tables 4(a), 4(b) and 4(c), where only one isotope (the one showing the best behaviour) has been selected for each target element. There were two groups of elements regarding the ordinate in origin: those elements with an ordinate in origin compatible with zero value within 99% CL and those ones with a valuebeingdifferentfromzeroatthesameCL.Figures1(a) and 1(b) show some examples for MT (noncompatible with zero and compatible with zero at 99% CL, resp.). The physical meaning of the slope is just the contribution to the instrumental signal (provided as 𝜇gL −1 in the aspired solution) per unit mass of original organic sample. Thus, in both cases, the concentration of the target analyte in the organic sample canbeobtainedfromtheslopeandthecommondilution factorusedforallsamples(resultsareshowninTables4(a), 4(b), and 4(c), with associated uncertainties arising from the error in the slope). The comparison of these values with certified/reference ones is also provided in Tables 4(a), 4(b), and 4(c), and it will be discussed further. The physical meaning of the ordinate in origin is just a contribution to the signal coming from matrix effects which are not present in the reagent blanks. Thus, these last include contributions from impurities in the reagent acids and water, as well as contamination throughout the analytical procedure. When a certain mass of organic sample is digested, even if the target analyte was absent, isobaric, polyatomic, and physical interferences could account to the background signal. It is worth to note that a direct quantification of the analyte using the total registered signal (corrected by reagent blank) and the dilution factor could misestimate the concentration value. A nonzero ordinate in origin could be likely lessened by using interference correction equations. Tables 4(a), 4(b), and 4(c) incorporate information of those elements showing nonzero ordinate in origin, and a brief discussion of the most relevant cases is presented in what follows. Isotope 51V has a negative ordinate in origin when using the correction equation (Table 2)whichbecomespositive when omitting it. This is related with the nonfully adapted (to instrument and matrix) values of the parameters in the correction equation. The slope was not affected by the use of the correction equation. A similar situation was found for 75As. Isotope 52Cr was used for element quantification (as recommended in the US-EPA 200.8 method), and its background was likely contributed by C and Ca in the matrix. Similarly, polyatomic interferences of 1H+59Co and 1H+ 64Zn could contribute to the observed background in the 60Ni and 65Cu signals, respectively. Isotopes 106Cd and 108Cd showed background contribution likely linked to Zn in the sample, but 111Cd was used for element quantification. Quantification of 133Cs was difficult due to the low concentration Table 3: Method detection limits (concentrations in mg kg−1) determined for laboratory blank solutions. Element m/z Equation#Procedure ABC Be 9 0.010 0.010 0.009 B 11 0.76 0.85 0.69 Al 27 2.9 4.7 2.0 Ti 47 2.5 1.4 3.5 V 51 (1) 0.12 0.69 0.44 V 51 0.12 N.R. 0.91 Cr 52 0.055 1.9 0.047 Mn 55 0.033 0.11 0.039 Fe 56 10.2 9.8 8.6 Co 59 0.012 0.013 0.024 Ni 60 0.15 0.38 0.12 Cu 63 0.33 0.71 0.44 Cu 65 0.50 0.27 0.17 Zn 66 1.4 0.61 0.36 As 75 0.020 N.R. 0.20 As 75 (2) 0.27 0.86 0.20 Se 82 (3) 0.87 1.4 0.89 Se 82 0.88 0.89 0.97 Sr 88 0.045 0.014 0.028 Mo 95 0.26 1.13 0.32 Ag 107 0.081 0.013 0.009 Cd 111 (4) 0.083 0.055 0.081 Sb 123 0.005 0.004 0.007 Cs 133 0.014 0.028 0.023 Ba 137 0.059 0.063 0.20 Tl 205 0.003 0.006 0.006 Pb 208 (5) 0.024 0.017 0.090 Th 232 0.005 0.003 0.007 U 238 0.002 N.Q. 0.003 Procedures: A (HNO3)andB(HNO 3+ HCl) use dilution factors of 0.6 g L−1, while 0.5 g L−1is applied for procedure C (HNO3+H 2O2). In the three cases, 0.5 g of dry sample was digested. For the variations of method A with different amount of sample, 𝑚𝑖(g), the corresponding MDLs have to be corrected by a factor of 0.5/𝑚𝑖. #Correction equations from Table 2. N.R.: not recommended; N.Q.: not quantified. and the interferences contributed by In, added as internal standard. Concerning the determination of analyte concentrations from the slope, the values reported in Tables 4(a), 4(b) and 4(c), were in reasonable good agreement with the certified/reference ones, but in some cases significant statistical differences were found. When plotting the determined slope versus certificated/reference values of concentrations for all the analytes (not shown), a linear relationship holds at 99% CL (𝑅2= 0.991,𝑛=38)withslope0.886 ± 0.014. This last provides a gross estimation of the digestion yield, but elemental yields depend on the element geochemistry. Thus, incomplete recoveries in the digestions were found 6ISRN Analytical Chemistry 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 00 0.2 0.4 0.6 0.8 1 Sample mass (g) Analyte concentration (𝜇gL−1) 60Ni 52Cr 106Cd (a) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 00 0.2 0.4 0.6 0.8 1 Sample mass (g) Analyte concentration (𝜇gL−1) 66Zn/137 208Pb 111Cd (b) Figure 1: Examples of linear regression for a selected group of elements measured in MT matrix: (a) elements with noncompatible with zero ordinate in the origin at 99% CL and (b) elements with compatible with zero ordinate in origin at 99% CL. See text for explanation. Analyte concentrations are reported as a mean value and standard deviation from the three analytical replicates. forFeandSrinMTandforAl,V,Cr,Fe,Cu,andSbin TL. Overestimations were identified for Se in MT (likely due to poor signal calibration in the low concentration range) and Co and Ni in TL. 82Se recoveries could also be affected by 81Br1H interference (MT has a Br concentration of 329 mg kg−1). 59Co is also affected by an interference with Ca (formation of species 43Ca16Oand42Ca17OH), present in TL in a percentage of 5.05%. Arunachalam et al. [38]havealso reported this overestimation in materials BCR CRM-141 and BCR CRM-142 digested by HNO3(118% and 110%, resp.) and by a mixture of HNO3+HCl+HF(127%and106%,resp.). 3.4. Procedure A with Scaling Masses: Direct Quantification through the Dilution Factor. Concentrations of target analytes can be directly quantified for each amount of digested organic sample by using the corresponding dilution factors. This procedure has been applied in its standard version, that is, the recorded signal is corrected only by subtraction of the reagent blank and applying, when appropriate, the interference correction equations. Detailed results will not be reported here, but a general discussion is presented below. The good linear relationship reported previously already ensures consistent results for the group of elements which are free of background contribution from matrix effect (those reported in Tables 4(a), 4(b), and 4(c) with compatible zero ordinate in origin and which will be referred hereafter as group “a”). For the complementary group (group “b”, with background contribution), it is expected that a direct quantification will misestimate the values of the concentrations. It has been found that digestion of low amount of sample leads to results with higher statistical dispersion. Results with very high associated uncertainties can pass a test of comparison against certified or reference values, but precision might be a target objective for the analyst. Thus, relative uncertainties have been estimated for each quantifiable analyte, and after, their averaged value and standard deviations were found for each digested mass. The resulting magnitude can be compared for the different masses ranging from 100 up to 900 mg. Results are shown in Figure 2 for MT and TL matrices. The smaller mass amounts (100 and 200 mg) produced overall higher relative uncertainties. A statistical test to compare the mean values has been conducted to detect significant differences among the direct determination and those previously found from the slope. In this way, the effect of incomplete recovery during the digestion procedure can be discarded. The total number of failures found in MT and TL matrices has been quantified for each digested mass and separately for “a” and “b” class analytes. Results are reported in Figure 3, given as a percentage of the total number of analyte quantifications involved in each case. As expected, most of the direct determinations for class “b” analytes failed, with a trend of decreasing for increasing amounts of digested samples. The percentage of failures for “a” class analytes is small and concentrated around the low masses range. Actually, they are likely linked to the statistical variabilitypreviouslyreportedinFigure 2. Class “b” elements should not be directly quantified without an appropriate treatment of physical and isobaric interferences due to matrix effects. Thus, the selection of the most suitable amount of mass to be digested should fulfil two criteria: precision and accuracy. The first one can be quantified by the associated relative uncertainties; meanwhile, for thesecondcriteria,thepercentageoffailuresincomparison ISRN Analytical Chemistry 7 Table 4: (a) Measured (from slope) and certified/reference concentrations (mg kg−1) for the target analytes in the SRM 2976 mussel tissue. (b) Measured (from slope) and certified/reference concentrations (mg kg−1)forthetargetanalytesintheSRM1549nonfatmilkpowder.(c) Measured (from slope) and certified/reference concentrations (mg kg−1) for the target analytes in the SRM 1573a tomato leaves. (a) Element 𝑚/𝑧 SRM 2976 mussel tissue From slope Certified/reference Stat. Be 9 N.L. B1122.5±1.0 Aa Al 27 140±11 R134 ±4 Ab-Ca Ti 49 15.02 ±0.57 Aa V511.39± 0.10 Ab Cr 52 0.324± 0.029 R0.50 ± 0.16 Ab-Ca Mn 55 37.1± 1.2 R33.0 ± 2.0 Aa-Ca Fe 56 112.3 ± 7.5 C171.0 ±4.9 Aa-Cb Co 59 0.644±0.021 R0.61 ± 0.02 Aa-Ca Ni 60 0.82 ± 0.04 R0.93 ± 0.12 Ab-Ca Cu 65 3.35 ± 0.17 C4.02 ± 0.33 Ab-Ca Zn 66 137±6 C137 ± 13 Aa-Ca As 75 14.6 ± 0.5 C13.3 ± 1.8 Aa-Ca Se 82 2.75± 0.23 C1.80 ± 0.15 Aa-Cb Sr 88 67.0 ± 2.4 R93 ±2 Aa-Cb Mo 98 0.412 ± 0.025 Ab Ag 107 N.L.; <MDL R 0.011± 0.005 Cd 111 0.938± 0.03 C0.82 ± 0.16 Aa-Ca Sb 123 N.L. Cs 133 N.L.; <MDL R 0.027± 0.001 Ba 137 0.65 Tl 205 N.L. Pb 208 1.19± 0.04 C1.19 ± 1.18 Aa-Ca Th2320.0123± 0.0035 R0.011 ± 0.002 Bb-Ca U2380.244± 0.008 Aa Statistical analysis: first (capital) refers to linear fit: A >99% CL, B >95% CL; C >90% CL. Second (lower case) refers to the ordinate in origin: not significantly different from zero at 99% CL; b, else. Third refers to the comparison between measured and certificated/reference values: Ca not statistically significant difference at 95% CL, Cb, else. For those reference values without associated uncertainties, relative errors of 20% have been assumed. N.L: nonpositive linear relationship; <MDL: below method detection limit; R: reference value; C: certified value. (b) Element 𝑚/𝑧 SRM 1549 nonfat milk powder From slope Certified/reference Stat. Be 9 N.L. B112.8± 0.7 Ba Al 27 N.L.; <MDL R 2.0 Ti 49 2.63 ± 0.17 Aa V51N.L. Cr 52 N.L.; <MDL C 0.0026± 0.0007 Mn 55 0.254± 0.010 C0.26± 0.06 Ab-Ca Fe 56 N.L.; <MDL C 1.78± 0.1 Co 59 <MDL R 0.0041 Ni 60 0.308 ± 0.027 Ab Cu 65 0.71 ± 0.29 C0.70 ± 0.1 Ca-Ca Zn 66 43.9 ± 1.4 C46.10 ± 2.2 Aa-Ca As 75 <MDL R 0.0019 Se 82 <MDL C 0.110 ± 0.010 8ISRN Analytical Chemistry (b) Continued. Element 𝑚/𝑧 SRM 1549 nonfat milk powder From slope Certified/reference Stat. Sr 88 3.68 ± 0.12 Aa Mo 98 0.350 ±0.017 R 0.34 Aa-Ca Ag 107 <MDL R <0.0003 Cd 111 <MDL C 0.0005± 0.0002 Sb 123 <MDL R 0.00027 Cs 133 N.L. Ba 137 0.87±0.04 Aa Tl 205 N.L.; <MDL Pb 208 <MDL C 0.019± 0.003 Th232N.L. U238N.L. Statistical analysis: first (capital) refers to linear fit: A >99% CL; B >95% CL; C >90% CL. Second (lower case) refers to the ordinate in origin: not significantly different from zero at 99% CL; b, else. Third refers to the comparison between measured and certificated/reference values: Ca not statistically significant difference at 95% CL, Cb, else. For those reference values without associated uncertainties, relative errors of 20% have been assumed. N.L: nonpositive linear relationship; <MDL: below method detection limit; R: reference value; C: certified value. (c) Element 𝑚/𝑧 SRM 1573a tomato leaves From slope Certified/reference Stat. Be 9 0.0274 ± 0.0016 Ab B1130.5 ± 1.1 Aa Al 27 543 ± 12 C598 ± 12 Aa-Cb Ti 49 22.6 ± 1.6 Aa V510.56 ± 0.05 C0.835 ±0.010 Ab-Cb Cr 52 1.51±0.06 C1.99± 0.06 Ab-Cb Mn 55 236 ± 9 C246 ± 8 Aa-Ca Fe 56 301± 8 C368 ± 7 Ab-Cb Co 59 0.756±0.022 C0.57 ± 0.02 Aa-Cb Ni 60 1.93± 0.10 C1.590 ± 0.07 Ab-Cb Cu 65 3.85±0.23 C4.70 ± 0.14 Aa-Cb Zn 66 29.0 ± 1.8 C30.90 ± 0.7 Aa-Ca As 75 0.063 ± 0.008 C0.112 ± 0.004 Ab-Cb Se 82 <MDL C 0.054± 0.003 Sr 88 78.2± 1.8 R85Aa-Ca Mo 98 0.400 ± 0.010 Aa Ag 107 0.016 ± 0.004 R 0.017 Aa-Ca Cd 111 1.56 ± 0.04 C1.52 ±0.04 Aa-Ca Sb 123 0.035 ± 0.002 C0.063 ± 0.006 Aa-Cb Cs 133 0.036 ± 0.005 R 0.053 Ab-Ca Ba 137 57.6 ±1.6 R63Aa-Ca Tl 205 0.0444± 0.0018 Aa Pb 208 0.594 ± 0.016 Aa ISRN Analytical Chemistry 9 (c) Continued. Element 𝑚/𝑧 SRM 1573a tomato leaves From slope Certified/reference Stat. Th2320.101± 0.003 R 0.120 Aa-Ca U2380.0298± 0.0015 R 0.035 Aa-Ca Statistical analysis: first (capital) refers to linear fit: A >99% CL; B >95% CL; C >90% CL. Second (lower case) refers to the ordinate in origin: not significantly different from zero at 99% CL; b, else. Third refers to the comparison between measured and certificated/reference values: Ca not statistically significant difference at 95% CL, Cb, else. For those reference values without associated uncertainties, relative errors of 20% have been assumed. N.L: nonpositive linear relationship; <MDL: below method detection limit; R: reference value; C: certified value. TL MT 103 102 101 100 10−1 Relative mean error (%) 0 0.2 0.4 0.6 0.8 1 Sample mass (g) Figure 2: Statistical variability estimated for each quantifiable analyte in matrices TL and MT. The resulting magnitude can be compared for the different masses ranging from 100 up to 900 mg (the smaller mass amounts produced overall higher relative uncertainties). against reference values can serve as a reasonable quantification. Thus, the following “figure of merit” has been defined: FM =𝑓 1(100− %failures)+𝑓 2⟨𝜀𝑟⟩ 𝜀𝑟(𝑚𝑖),(1) where 𝑓1and 𝑓2are user-defined weighting factors, 𝜀𝑟(𝑚𝑖) istheaveragedrelativeuncertaintyfoundformass𝑚𝑖(from Figure 2), and ⟨𝜀𝑟⟩is the averaged value for all sample masses. Figure 3 shows this “figure of merit” for 𝑓1=𝑓 2=50.The best mass amount to be used in the digestion procedure is foundtobearound600mg,whichcorrespondswithasample mass to acid volume ratio of 60 mg mL−1.Slightchangesin the values of the weighting factors 𝑓1and 𝑓2do not affect to the final result. This sample mass to acid volume ratio is similar to those reported elsewhere. For example, by Sucharov´ a and Suchara [1]usearelationmasssampletoacidratioof 36 and 72 mg mL−1 (depending on the concentration of Si present in the samples) to determine the concentration of 120 100 80 60 40 20 00.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Sample mass (g) a (%) b (%) F-M (50%, 50%) Fails in test of mean values (%) Figure 3: Percentages of direct determinations from the dilution factor with statistically significant differences (at 95% CL) with respect to the value found from the slope (see Table 4 and Figures 1(a) and 1(b))inMTandTLmatrices).Classes“a”and“b”refer to isotopes with an ordinate in origin statistically nondifferent and statistically different from zero at 99% CL, respectively (see text for explanation). The “figure of merit” (as defined in the text) pursuits the digested mass with the best compromise between precision and accuracy. 36 elements in different plant reference materials by ICP-MS after microwave digestions assisted by three different types of mixtures. Gonz´ alez et al. [39] use a ratio of 67 to determine by ICP-OES the concentration of 7 elements in plant and animal samples after a microwave-assisted digestion with also nitric acid and hydrogen peroxide. Rodushkin et al. [40]reported a ratio of 50 for a microwave digestion with nitric acid and hydrogen peroxide prior to the determination of 16 elements in the certified reference material SRM 1547 (peach leaves) by ICP-AES, and ICP-SMS is reported. Finally, Sastre et al. [15]usedaratioof66inanaciddigestionwithnitricacid for the determination of Cd, Cu, Pb, and Zn in a wide range of environmental samples (covering sediment, soil, sewage sludge, and plant matrices) by ICP-MS.