Electrochemical preconcentration coupled with spectroscopic techniques for trace lead analysis in olive oils

Journal Pre-proof Electrochemical preconcentration coupled with spectroscopic techniques for trace lead analysis in olive oils M. Antonietta Baldo, Angela M. Stortini, Paolo Oliveri, Riccardo Leardi, Ligia M. Moretto, Paolo Ugo PII:

S0039-9140(19)31300-1

DOI:

https://doi.org/10.1016/j.talanta.2019.120667

Reference:

TAL 120667

To appear in:

Talanta

Received Date: 2 October 2019 Revised Date:

16 December 2019

Accepted Date: 20 December 2019

Please cite this article as: M.A. Baldo, A.M. Stortini, P. Oliveri, R. Leardi, L.M. Moretto, P. Ugo, Electrochemical preconcentration coupled with spectroscopic techniques for trace lead analysis in olive oils, Talanta (2020), doi: https://doi.org/10.1016/j.talanta.2019.120667. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Electrochemical Preconcentration coupled with Spectroscopic Techniques for Trace Lead Analysis in Olive Oils

M. Antonietta Baldoa, Angela M. Stortinia, Paolo Oliveri b, Riccardo Leardi b, Ligia M. Morettoa, Paolo Ugoa a

Department of Molecular Science and Nanosystems, Ca’ Foscari University of Venice, Via Torino 155, I-30172 Venezia Mestre, Italy b

Department of Pharmacy, University of Genova, Viale Cembrano 4, 16148 Genova, Italy

* Corresponding author, e-mail: [email protected]; phone: 0039-041 2348646

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Abstract In this paper we present a novel combined electrochemical-spectroscopic approach suitable to monitor trace levels of heavy metals directly in edible oils.

The method is based on the electrochemical

preconcentration/extraction of the analyte from the tested real matrix by cathodic deposition onto a Pt working electrode, then transfer and anodic re-oxidation of the metallic deposit to a “clean” aqueous solution, suitable for the subsequent spectroscopic analysis. The procedure has been here focused to the determination of lead in extra virgin olive oil (EVOO), performed by applying ICP-QMS or GFAAS techniques. To this aim, the EVOO samples were mixed with proper amounts of the room temperature ionic liquid (RTIL) [P14,6,6,6]+[NTf2]-, in order to obtain a non-aqueous supporting electrolyte suitable for the electrodeposition process. The feasibility and performance of the analytical strategy were at first tested in standard solutions of Pb(II) in RTIL, produced by anodic dissolution of lead in the RTIL, as well as in olive oil samples mixed with 0.5 M RTIL and spiked with known amounts of Pb(II). The optimization of the electrochemical parameters was achieved by applying a D-Optimal Design, properly set up to optimise the efficiency of the deposition and re-oxidation steps, quantitative recovery and measurement time. Finally, the analytical procedure was applied to the determination of Pb content in some Italian EVOOs, without any need of performing mineralization pretreatments. Data obtained with the proposed procedure satisfactorily agree with those achieved by ICP-QMS analysis after microwave digestion, being differences between the two approaches within 10%, with the advantage of reducing to half the pretreatment time, operating at room temperature and avoiding the use of aggressive solvents.

Keywords: Electrochemical preconcentration; trace lead; spectroscopic analysis; olive oil; ionic liquid; experimental design.

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1. Introduction Trace amounts of heavy metals can be present in olive oil because of contaminations originating from different sources, such as soil and fertilizers, production or storage procedures, or exposition of the olive plants to vehicular and industrial emissions [1-5]. The quality of the oil is strictly related to the concentration of metal species present in the final product, since trace elements like Cu, Fe, Ni and Zn may catalyse reactions that promote the oxidative degradation of the edible oil. Moreover, other metals, such as Pb, Cd or Hg, are potentially toxic for human consumption. [4, 5]. Thus, the determination of trace metals content in edible oil is crucial for assessing the quality, both from health and economic points of view. However, this analytical goal constitutes a challenging task, due to the very low concentration levels of these analytes, as well as to the high complexity of the organic matrix of vegetable oils. In particular, the analysis of metal ions in oil using conventional analytical instrumental techniques requires the application of complex and time-consuming pre-treatment steps, which are a potential source of contamination of the sample or loss of analyte, possibly reflected in scarce accuracy and precision. By far, atomic spectroscopic techniques, including graphite furnace atomic absorption spectrometry (GFAAS) [4, 6-14], flame atomic absorption spectrometry (FAAS) [4,8,15,16], inductively coupled plasma atomic emission spectrometry (ICP-AES) [6-8] and inductively coupled plasma mass spectrometry (ICP-MS) [3, 5, 17, 18]

are the

techniques most commonly used for the analysis of trace metal in edible oils. A few papers have been also reported on the use of electroanalytical methods, such as derivative potentiometric [1, 2, 19] and voltammetric stripping analysis [20] using Hg-based electrodes. Whatever the analytical technique used, sample preparation is a critical step in the whole analytical process. Various pre-treatment procedures have been proposed and applied to the analysis of edible oils, most of them including dry or wet ashing [1, 8, 10], microwave-assisted acid digestion [3-8, 17, 18,20], dilution with organic solvents [8,9,21], liquid-liquid extraction [10,11,13,19,22,23], emulsion or microemulsion preparations [8,16]. All approaches show some advantages, but also several drawbacks, especially in terms of time-consuming procedures, aggressive conditions, use of hazardous solvents even in large amounts, low recovery rates, contamination risks [10-13,22]. Accordingly, improvements and optimisation of the sample pre-treatment step are highly demanded. With the goal of developing a fast and reliable analytical approach, specifically suitable to monitor trace levels of heavy metals directly in edible oils, in the present study a novel strategy is presented, which combines electrochemical preconcentration with spectroscopic analysis, focusing on the determination of lead in extra-virgin olive oil as a case study. In the literature, the introduction of an electrochemical preconcentration (EC) step as an auxiliary tool for subsequent ICP-MS or ICP-AES determination of trace metals in complex aqueous matrices, has been proposed [24-27]. For instance, such an approach has been used to detect As (III) and Se (IV) [25], Cr (VI) and V(V) [26], Cu and Cd [27] in environmental and biological samples, and Hg in process and lagoon waters [24]. 3

The alternative approach developed here is based on the use of a platinum spiral electrode as preconcentration/extraction tool, where the metal species of interest, namely Pb, is at first electrochemically deposited. After suitable medium exchange, the deposited metal is anodically redissolved in a “clean” aqueous solution, suitable for ICP-MS or GFAAS analysis, or other kind of technique. In order to perform the electrochemical preconcentration of the metal from a complex and low-conductive matrix such as olive oil, the room temperature ionic liquid (RTIL) tri-hexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl)imide [P14,6,6,6]+[NTf2]-, which is soluble in vegetable oils, was used as the supporting electrolyte [28-31]. For evaluating quantitatively the performances of the procedure, blank oil samples were spiked with known amounts of Pb in RTIL. For this purpose, standard solutions of lead in [P14,6,6,6]+[NTf2]- were produced by galvanostatic anodic dissolution directly in the RTIL medium of a lead anode, using the electrochemical procedure recently developed in our laboratory [32]. Optimisation of the electrochemical parameters ruling deposition and re-oxidation of lead onto and from the Pt working electrode was performed by resorting to D-Optimal design [33-35]. A key feature of DOptimal design – exploited in the present study – is the possibility of considering experiments previously performed, obtaining a good design by adding to them a relatively small number of new experiments [36]. Finally, the selected analytical protocol was applied to the combined electrochemical preconcentration and GF-AAS or ICP-QMS determination of the Pb content in real samples of Italian extra-virgin olive oil, without any mineralization of the matrix.

2. Experimental 2.1 Reagents and samples The RTIL [P14,6,6,6]+[NTf2]- (assay ≥ 95.0 %) and the lead standard solution for AAS (TraceCERT 1 mg mL-1 in 1M HNO3) were purchased from Sigma-Aldrich (Milan, Italy). HNO3 (67-69 w/w %, Romil® Suprapur) was obtained from Delchimica (Napoli, Italy). All chemicals were used as supplied by the manufacturer. According to the technical sheet, the water content of [P14,6,6,6]+[NTf2]- was lower than 1000 mg L-1. To avoid any further contamination with water, the RTIL was kept in a desiccator after opening. The oil samples examined in this study were extra-virgin olive oil (EVOO) commercially available, produced in Italy. They were stored at room temperature under dark conditions (in amber bottles), until sample preparation and analysis. For the development and validation of the analytical procedure, lead standard solutions in pure RTIL were home-produced by electrochemical oxidation of a bar of Pb (size: 0.5x0.2x1.5 cm, section area: 0.15 cm2), used as sacrificial anode, as reported in a previous study [32].

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When required, pure nitrogen (≥ 99.9% SIAD, Bergamo, Italy) was used to remove oxygen from the solution. All materials used for samples treatment were cleaned preliminarily with Contrad 2000 detergent (Z.E.U.S., Bolzano) and HNO3 (Romil® Suprapur), then accurately rinsed with MilliQ (Merck Millipore, Darmstadt, Germany) ultrapure water (resistivity 18.2 MΩ cm-1). To validate the data obtained from ICP-QMS measurements, the multielemental reference standard Certipur® Merck (Milan, Italy), containing 100 (±5) μg g-1of Pb(II) in paraffin oil, was employed.

2.2. Instrumentation and procedures 2.2.1. Electrochemical measurements Electrochemical experiments were carried out at room temperature (21±1°C) by using an electrochemical workstation CHI660 (CH Instruments, USA) controlled via PC. The potentiostatic electrochemical deposition of the metal ion from the tested matrix was performed by using a homemade 5 mL polypropylene cell, which was assembled in an undivided three-electrode cell configuration equipped with a Pt coil (1 cm length, 0.5 cm coil diameter and 0.4 mm wire thickness) working electrode (WE), a Pt plate counter electrode (CE) and an Ag wire (1 cm length and 1 mm diameter) pseudo-reference electrode (AgPsRE). Before use, the Pt coil working electrode was carefully polished by dipping it in 10 mL of Suprapur HNO3 (67-69%) for about 10-15 min, then accurately rinsed with MilliQ purified water in ultrasound bath for 10 min. The potential of the Ag pseudo-reference in the non-aqueous medium was checked frequently by recording the half-wave potential (E1/2) of the ferrocene/ferricinium (Fc/Fc+) couple taken as an internal redox standard [37], which resulted +0.195 (±0.005) V. Electrochemical deposition of the analyte was performed in 2 mL volume samples at room temperature (i.e., 21± 1°C), previously deoxygenated by purging with N2, dried by passing through concentrated H2SO4, and under stirring for about 45 min. For the following potentiostatic anodic re-oxidation of the metal deposit, the three electrodes were then transferred, after cleaning carefully the electrode tips with few drops of acetone, to a 5 mL homemade polypropylene cell containing 2 mL volume sample of 10% HNO3 solution in MilliQ purified water.

2.2.2. Spectroscopic analysis Lead content in the solutions investigated was quantified using an Agilent 7500 I Inductively Coupled Plasma-Quadrupole Mass Spectrometer (ICP-QMS), or a dual beam Atomic Absorption Spectrophotometer Varian SpectrAA 250 Plus equipped with an Agilent GTA-96 graphite furnace atomizer GFAAS (Agilent, Santa Clara, United States).

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For ICP-QMS measurements, the plasma conditions were: RF Power: 1500 W, carrier Ar flow rate: 1.2 L min1

, torch horizontal alignment: 0.6 mm, torch vertical alignment: -0.4 mm, sampling depth: 8 mm,

instrument sampler cone in Nickel. Sample introduction during analyses was carried out by a Cetac ASX500autosampler. Each value obtained was the average of seven readings per sample (with RSD < 3%) and concentration data were computed after subtraction of the blank value. For obtaining the calibration plot, standard solutions at Pb(II) concentration ranging from 0.02 up to 20 ng g-1 were prepared from the multielemental standard ICUS 1675, 10 mg/mL in 5%HNO3 (ULTRA Scientific Italia S.r.l., Bologna, Italy) with lead at the certified concentration. A spike of Tl (50 ppb) was added in each vessel to assess the recovery; values ranging between 94-97% were found. For GFAAS measurements, argon was used as the inert gas, with the following operating conditions: Ar flow rate: 0.3 L min-1, sample volume: 24 µL, no addition of matrix modifier. The heating program temperature steps for the graphite furnace were set as reported in Table 1. Data obtained by GFAAS measurements were the average of ten readings per sample (with RSD < 3%), after subtraction of blank values. In this case, for the calibration plot standard Pb(II) solutions at concentration ranging between 0.4 and 10 ng g-1 were prepared by dilution from TraceCERT Lead standard for AAS, 1 mg mL-1 in 1M HNO3. Quantitative analysis of the Pb content by ICP-QMS and GFAAS techniques was performed by means of calibration plots obtained in 10% HNO3 aqueous solutions. Linear regression analysis of the experimental data provided: i) Count/CPS (Pb 207) = 4.4643x104 CPb (ng g-1) + 7.580 x102, R2 =0.9999, linear range 0.02 – 20 ng g-1 and ii) Abs = 7.55 x10-2 CPb (ng g-1) + 5.42 x10-2, R2 =0.9991, linear range 0.4 – 10 ng g-1, for ICPQMS and GFAAS measurements, respectively. The limit of detection (LOD) was calculated as: LOD = 3 sb/m, where sb is the standard deviation of the blank (evaluated with 10 measurement replicates) and m is the slope of the calibration line (sensitivity). Analogously, the limit of quantification (LOQ) was calculated as: LOQ = 10 sb/m.

2.2.3. SEM – EDS analysis To control and map the amount of lead deposited electrochemically on the Pt coil electrode, Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS)analyses were performed using a TM3000 Hitachi Tabletop microscope equipped with an Oxford EDS system and SwiftED software, version 1.7 (Oxford Instruments Analytical Ltd.). For SEM signal acquisition, the following parameters were set: time acquisition: 30 s, process time: 5 s, beam in accelerating voltage: 15 kV, pressure in charge-up reduction mode.

2.2.4. Preparation, standardization and spiking with Lead stock solution in RTIL

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Lead stock solutions containing 20 μg g-1 nominal concentration of Pb(II) in [P14,6,6,6]+[NTf2]-were prepared electrochemically, as described in a previous study [32], by galvanostatic oxidation of a Pb anode in 2 mL of RTIL, equilibrated at room temperature, previously purged with N2 for about 45 min and kept under stirring at 150 rpm, by applying a current of 1x10-4 A (6.7x10-4 A cm-1) and an oxidation time (tox) of 400 s. Known volumes of stock solutions were spiked to oil or oil/RTIL mixtures to evaluate the recovery of the analytical procedure i.e. electrochemical deposition, medium exchange, reoxidation and spectroscopic analysis. The experimental Pb(II) concentration value was calculated on the basis of the experimental charge, Qexp, consumed during the electrochemical dissolution step. Then, it was validated by comparison with data determined on the same samples by ICP-QMS analysis after microwave (MW) digestion. This was performed applying the following procedure specifically set for mineralising the complex organic matrix of the RTIL medium [32]: 0.5 g of each sample (weighted with precision of ± 0.1 mg) was diluted with 8 mL Suprapur HNO3 (67-69%), then digested in a microwave unit Ethos1-Milestone® (Bergamo, Italy) by applying a mild heating controlled program from room temperature up to 180 °C, and fixing the power at 1500 W [32]. After digestion, each sample was recovered from the Teflon® vessel and diluted ten times with ultrapure MilliQ water.

2.2.5. Preparation of the olive oil/RTIL mixtures Prior to the electrochemical metal deposition, the olive oil/[P14,6,6,6]+[NTf2]- mixtures were prepared by mixing accurately weighed amounts (with precision of 0.1 mg) of the two liquids inside a 2 mL glass vial. On the basis of previous findings [28-30], the suitable ratio of olive oil and RTIL was 50/50 (% w/w), which corresponds to a concentration of 0.5 M RTIL in oil. To allow a fast homogenisation between the two liquids, they were mixed by an Advanced Vortex Mixer (Velp Scientifica, Italy) for 3 min, and then left to equilibrate for about 5 min, before performing the measurements. By this way, a visibly clear homogeneous solution, with absence of turbidity, was obtained. The same procedure was also employed to homogenise the olive oil/RTIL mixture after it was spiked with different amounts of the Pb/RTIL stock standard solution, prepared electrochemically as described in the previous paragraph.

2.2.6. Experimental Design In order to optimise the electrochemical deposition and re-oxidation parameters, and estimate the relevant recovery, the effect of the following four factors was studied: 1) Deposition time, tdep (range = 5 ÷ 60 min); 2) Oxidation time, tox ,( range = 2 ÷ 16 min); 3)Deposition potential, E

dep

(E

dep

= -2.0, -3.5, -5.0 V) ; 4)

Oxidation potential, E ox (E ox = +0.5, +1.0, +1.5 V). Recovery of Pb (%) was chosen as response.

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A D-Optimal design was applied to expand a first series of experiments– preliminary performed – with new experiments purposely designed, with the aim of building an efficient model for studying the effects of factors and their interactions. The new experiments are chosen by the algorithm, among all the possible combinations of the levels of the different factors, so as to meet the D-Optimality criterion, which consists in the maximisation of the determinant of the information matrix (X’X) [33, 36]. In this way, the minimum number of experiments to build an informative model is chosen. Experimental matrices and design models were computed by the CAT (Chemometric Agile Tool) software, based on the R platform (The R Foundation for Statistical Computing, Vienna, Austria) [38].

3. Results and discussion 3.1. Development of the analytical procedure The feasibility of the electrochemical-spectroscopic approach proposed here was investigated by performing experiments both in diluted standard solutions of Pb(II) in pure RTIL and in oil/0.5M RTIL mixtures spiked with Pb(II), and adopting the following analytical procedure: ● Potentiostatic electrochemical deposition of the metal ion from the tested non-aqueous sample onto the Pt coil electrode, by applying a suitable negative deposition potential, Edep, and deposition time, tdep. ● Transfer of the Pt coil electrode from the RTIL or oil/RTIL tested medium to another cell containing a small quantity of pure nitric acid diluted in water (see Fig.1). ● Anodic re-oxidation of the Pb deposit by applying a constant oxidation potential, Eox, for a selected oxidation time, tox. ● ICP-QMS and/or GFAAS analysis of the collected acid solutions.

Electrochemical deposition of lead from the tested non-aqueous matrix onto the Pt coil electrode Preliminarily, the possibility of obtaining an effective electrodeposition onto the Pt coil electrode of the Pb ions dissolved in the non-aqueous matrices under investigation was tested in deaerated Pb(II) /RTIL solutions at a relatively high concentration level, i.e. with CPb(II) ranging between 50 and 1000 ng g-1, which were obtained by dilution of the standard Pb(II) stock solution in RTIL (20 ug g-1) prepared by the procedure previously described [32]. Analogously, the electrodeposition procedure was tested also in oil/RTIL mixtures. Fig. 2 shows a typical chronoamperometric response recorded in a 2 mL volume of oil/RTIL sample enriched with CPb(II) =150 ng g-1, by applying a very negative Edep value, namely Edep = -5.0 V, for a quite long deposition time, i.e. tdep = 30 min. After an initial jump related to double layer charging, the cathodic current increased with time, from approximately 1.75x10-4 A at about t=2 min to 1.95x10-4 A at t=30 min. This behaviour agrees with a growth of the electrode area with time, so indicating the deposition 8

of a conducting deposit onto the surface of the Pt coil electrode. This typical transient behaviour was employed to check the formation of the metallic deposition onto the electrode surface during the preconcentration step. The occurrence of an effective electrodeposition of lead from the solution to the Pt coil electrode was also verified and controlled by performing SEM-EDS analysis on the Pt coil both before and after applying the metal preconcentration procedure. A typical SEM image of the coil electrode carefully cleaned with hot concentrated nitric acid is shown in Fig.3a, which evidences the clean and smooth surface of the platinum substrate before Pb deposition. Instead, the EDS elemental map obtained after the electrodeposition procedure clearly shows, as green spots (see Fig.3b), the metallic Pb deposited homogeneously on the whole coil surface. Moreover, relevant EDS spectrum recorded after the electrodeposition step evidenced the typical Pb signals, in the range 1.82 - 2.65 KeV and 9.18 - 15.10 KeV, i.e. at energy values characteristics for M and L shells of lead [39]. Re-oxidation of the Pb deposit after medium exchange After the metal preconcentration step, the three-electrode assembly was transferred from the nonaqueous matrix under investigation to a 10%HNO3 aqueous solution. At a first attempt, a set of re-oxidation measurements was carried out by applying a quite positive constant oxidation potential, i.e. Eox = +1.5 V, for a relatively short oxidation time, i.e. tox= 1min, starting from three RTIL solutions containing CPb(II) = 50, 150 and 500 ng g-1, respectively. The procedure was then repeated by applying the same deposition protocol in the three Pb/RTIL solutions but re-oxidising at Eox = +1.5 V for tox= 6 and 10 min. This in order to have a first estimate of the occurrence and efficiency of the anodic reoxidation of the Pb amount deposited on the coil to the acidic aqueous medium, by applying these relatively short oxidation times. The so-collected acid solutions were then analysed by ICP-QMS. Relevant results are listed in Table 2. From these data, it can be observed that under such experimental conditions, recovery values in the range 40-55 % were found after applying the re-oxidation step for only 1 min. By increasing tox up to 10 min, while keeping constant the other electrochemical parameters, the recovery values increased to 72-75 %. Thus, as expected, the efficiency of the re-oxidation step increased with the oxidation time. However, it is worth pointing out that a totally exhaustive metal re-oxidation from the Pt coil is not strictly required; instead, a reliable calibration signal-concentration and accurate recovery estimate are needed, in order to exactly define the quantitative correlation between deposited and stripped metal, as well as to determine correctly the analyte concentration in the oil matrix [40] .

Preliminary experiments In order to gain preliminary information useful for setting a proper experimental design, a series of experiments was run, starting from an oil/0.5M RTIL mixture added with 50 ng g-1 Pb(II), by varying tdep 9

between 5 and 60 min, and keeping the other EC parameters constant as follows: Edep = -5.0 V, Eox = +1.5 V, tox = 10 min. The lead content in the aqueous sample collected after the re-oxidation step was then quantified by both ICP-QMS and GFAAS techniques. Table 3 collects the relevant outcomes; moreover, Fig.4 illustrates the trend obtained for Pb recovery (%) from both ICP-QMS (red points) and GFAAS (blue points) data, with increasing tdep. The comparison between the Pb(II) ion content determined by applying ICP-QMS and GFAAS detections (Table 3) shows the very good agreement between relevant data found by the two spectroscopic techniques, being the relative percentage error (RE %) within 5%.

3.2. Optimisation of the electrochemical deposition/re-oxidation parameters Experimental Design A first series of 6 experiments was performed in the development stages of the analytical protocol, empirically varying tdep and tox. With the aim of keeping these preliminary experiments for the building of the final model, a D-Optimal design by addition was applied. This was performed by considering the following four factors: 1) tdep,in the range = 5 ÷ 60 min; 2) tox, in the range = 2 ÷ 16 min; 3) Edep in the range = -2.0÷ -5.0 V ; 4)Eox in the range = +0.5 ÷ +1.5 V. D-Optimal design added 12 experiments – representatively exploring the variability of the four factors – to the 6 experiments already performed. The new experiments were performed in a random order, and the Pb(II) ions content in the matrix analysed was kept constant at 50 ng g-1. The experimental plan so obtained, with the sequence of values applied for the four electrochemical parameters, is presented in Table 4 (columns 2-5), in which the 6 preliminary experiments are reported as the first rows of the matrix (identified by Roman numbers). To estimate the precision of the procedure, in the experimental plan three replicates were also included; among these, it has been decided to replicate experiment IV, this in order to evaluate a possible block effect. This experimental matrix, made overall by 21 experiments, allowed to estimate the coefficients of the model with a sufficient quality (the highest Inflation Factor was 3.24) and with 6 degrees of freedom. The corresponding values of the X variables range-scaled between -1 and +1 [41], and the measured experimental values of response Y, i.e. Pb Recovery (%), are collected in Table 4, columns 6-10. The model, obtained by means Multiple Linear Regression, was the following: Y = 9.3 + 10.8 X1 + 15.5 X2 - 6.7 X3 + 13.4 X4 - 9.5 X1X2 – 4.8 X1X3 + 6.2 X1X4 + 6.5 X2X3 + 9.2 X2X4 - 6.6 X3X4 – 5.5 X12 + 8.6 X22 + 18.8 X32 + 10.2 X42

(1)

The model explains 77.7% of variance in fitting, with a standard deviation of the residuals of 10.1. It is therefore evident that the model cannot be used for quantitative predictions, but it is sufficiently good to allow a qualitative analysis of the effect of factors and their interactions. 10

By analysing the model coefficients (see eq.1), it can be noticed that the most relevant ones are the linear terms, the interactions X1-X2 (deposition and re-oxidation times) and X2-X4 (re-oxidation time and potential), and the quadratic term of X3 (deposition potential). Since all the variables are involved in relevant higher terms it is not possible to interpret their effect directly from the equation of the model. Fig. 5 a-b show the response surface on the plane X1 vs. X2 (Fig.5a) and the related contour plot (Fig. 5b), obtained by setting X3 = -1 (i.e. Edep=-5V) and X4 = +1 (Eox = +1.5 V). It can be seen that, as expected, an increase of both times produces an increase of the response, and that for both variables the effect is larger when the other variable is at low level. Moreover, the plot in Fig. 5b clearly evidences that by applying the longest pre-treatment time here considered, i.e. deposition time of 60 min and oxidation time of 16 min, a recovery value higher than 95% could be achieved. However, thinking to the practical applicability of the method, these conditions represent a relatively time-consuming procedure. Taking into account that a quite satisfactory recovery (around 70%) can be obtained with deposition time of only 30 min (-0.09 coded value) and an oxidation time of 10 min (+0.14 coded value), these settings were considered as a good compromise between a good recovery and a relatively short analysis time. Indeed, these conditions correspond to those of experiment V. Thus, according to IUPAC recommendations concerning correction-for-recovery procedures for the case of recovery values significantly < 100% [40], the experimental results were corrected for the relevant recovery value, in order to obtain the final analytical datum (see below).

3.4. Examples of application to Pb analysis in EVOOs The applicability of the proposed electrochemical/spectroscopic strategy for determining the lead content in edible oils, without any mineralization pre-treatment of the samples, was assessed by analysing three commercial Italian EVOOs. To this aim, the oil samples were preliminarily mixed with a suitable amount of [P14,6,6,6]+[NTf2]- to obtain a 0.5 M RTIL concentration in the mixture; then, the metal ions were preconcentrated and extracted from the EVOO mixtures to aqueous acid solutions by applying the electrochemical procedure investigated here. The Pb content in the collected acid solutions was then the quantified by GFAAS analysis. Data found by this approach in the tested EVOOs samples, corrected by applying a mean recovery value of 72.5 (± 3.0%) to the raw experimental data, are listed in Table 5. For comparison, the same oil/RTIL mixtures were also analysed by applying the previously reported [32]. MW acid digestion procedure for mineralising the organic matrix, followed by ICP-QMS analysis. Note that the reliability of the MW method was validated by its application to the analysis of a multi-elemental certified reference standard, 100 (± 5%) µg g-1 in paraffin oil [32]. The comparison between data found, listed in Table 5, indicates the good agreement between the two analytical approaches, with differences < 10%, so confirming also the reliability of the correction-forrecovery procedure [40] here adopted. Finally, it can be noted that concentration values determined for 11

the Pb content in the samples of Italian EVOOs here analysed correspond to the lowest levels of average Pb concentrations in EVOOs reported in the literature [2, 4-7, 10-13, 17, 20], which typically ranges from < 0.8 ng g-1 to approximately 100 ng g-1. Note that the maximum level acceptable for the Pb content in EVOOs established by the European Union Commission in Regulation N° 1881/2006 is 100 ng g-1 [42].

4. Conclusions This work demonstrates the possibility to exploit electrochemistry as efficient preconcentration tool to simplify significantly the analytical procedures required for the spectroscopic analysis of heavy metals in olive oil samples. The here proposed analytical procedure is based on the electrochemical reduction of metal ions in non-aqueous media composed by mixtures of the oil sample with a suitable RTIL. The inert electrode on which the analyte is deposited acts as a sort of preconcentration/extraction microdevice. The use of an experimental design method, such as the D-Optimal design used in this study, allowed the optimization of the experimental conditions, finally leading to an electrochemical pre-treatment procedure able to reduce to half the pretreatment time (from about 80 min to 40 min), furnishing analytical results comparable with the time consuming microwave digestion. Moreover, with respect to microwave digestion, the electrochemical procedure allows to operate at room temperature, with low energy consumption and reducing dramatically the use of aggressive reagents, so eliminating the production of possibly toxic wastes. The procedure has been successfully applied here to the case of the determination of lead in extra virgin olive oil, however it opens the way to wider applications, for the determination in nonaqueous matrices of trace heavy metals or other toxic elements (e.g. As) that can be electrochemically reduced to an elemental solid phase. This is a problem often found when analyzing food products, however it extends to a large variety of samples as, for instance, those of interest in oil or gasoline industry. Finally, we wish to point out that the preconcentration method here proposed can be combined practically with any analytical technique of interest, not only to the AAS and ICP-MS spectroscopic techniques applied here.

5. Acknowledgements This research was initially supported by MIUR (Rome, Italy), project PRIN 2010AXENJ8. Partial funding from Programma Operativo Regionale (POR) by Fondo Europeo di Sviluppo Regionale (FESR) 2014–2020, "Safe, Smart, Sustainable Food for Health (3S_4H)" is acknowledged. We also thank Ms. Lorena Gobbo from the Department of Molecular Science and Nanosystems, Ca’ Foscari University of Venice, for kindly performing GFAAS measurements of the samples.

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Figure captions

Fig.1 Schematized assembly of the proposed electrochemical-spectroscopic procedure for trace lead detection in olive oils. WE: Pt coil working electrode; RE: Ag wire pseudo-reference; CE: Pt counter electrode.

Fig.2 Typical chronoamperometric response recorded during the electrodeposition of lead onto the Pt coil electrode. Matrix: oil/0.5M RTIL mixture added with CPb(II) = 150 ng g-1 ; Edep = -5.0 V; tdep = 30 min.

Fig.3 SEM image of Pt coil before the Pb electrodeposition procedure (a) and relevant EDS elemental map (green spots) of electrodeposited Pb (b). Resolution: 296 X 192 pixels; acquisition time: 60 s; accelerating voltage: 15 kV; process time: 5.

Fig.4 Pb Recovery (%) values obtained with increasing tdep, from ICP-QMS (

) and GFAAS (

) data.

Sample: oil/0.5M RTIL mixture with CPb(II) = 50 ng g-1 ; Edep = -5.0 V; Eox = +1.5 V; tox = 10 min.

Fig.5 Response (Y) predicted by the experimental design model as a function of tdep (X1) and tox (X2), while keeping Edep (X3) = -5.0 V and Eox (X4) = 1.5 V. Surface (a) and contour plot (b).

Table 1- Experimental conditions for graphite furnace heating program

Heating program a

Drying

Ashing

Atomization

a

1

Temperature (°C) 85

Hold time (sec) 5

Argon flow (L/min) 0.3

2

95

40

0.3

no

3

120

10

0.3

no

4

400

5

0.3

no

5

400

1

0.3

no

6

400

2

0

no

7

2100

1

0

yes

8

2100

2

0

yes

9

2100

2

0.3

no

Step

: sample volume = 24 µL; no matrix modifier

Reading no

Table 2 – Pb(II) ions content determined after medium exchange by applying

different re-oxidation times nominal CPb(II) (ng g-1) in RTIL solutions

50

150

500

tox (min)

CPb, ICP-QMS (ng g-1) found in acid solutions

Recovery Pb (%) *

1

20.01

40.0

6

30.51

61. 2

10

36.2

72.4

1

83.20

55.5

6

95.33

63.6

10

112.8

75.2

1

235.1

47.0

6

295.7

59.1

10

372.5

74.5

* Recovery Pb % = CPb determined/CPb added x100; Electrochemical parameters: Edep = -5.0 V, tdep = 30 min; Eox = +1.5 V.

Table 3- Pb(II) ions content determined, after medium exchange, by applying

different tdep during the EC deposition step

a b

Tdep (min)

CPb, ICP-QMS (ng g-1)a

CPb, GF-AAS (ng g-1)b

RE %c

5

19.2 (0.8 %)

20.1 (1.6%)

4.7

10

23.9 (0.6%)

24.8 (1.8%)

3.8

15

27.2 (0.9%)

28.0 (2.0%)

2.9

30

35.8 (1.1%)

36.2 (1.5%)

1.1

60

38.0 (1.0%)

38.7 (1.2%)

1.8

:RSD % in parenthesis calculated from 7 readings for sample. LOD =0.019 ng g-1; LOQ=0.063 ng g-1 -1

: RSD % in parenthesis calculated from 10 readings for sample. LOD: 0.12 ng g ; LOQ= 0.40 ng g : Relative percentage error, RE% = (CPb, GF-AAS - CPb, ICP-QMS)/ CPb, ICP-QMS x100

c

-1

Table 4 – Experimental matrix reporting the 21 experiments performed, with the

sequence of values applied for the four electrochemical parameters and their correspondent range-scaled X variables, and the measured experimental values of the Y response (Pb recovery %)

Experiment tdep(min) tox (min) Edep(V) Eox (V) I 30 2 -5.0 1.5 5 II 10 -5.0 1.5 III 10 10 -5.0 1.5 IV 15 10 -5.0 1.5 V 30 10 -5.0 1.5 VI 60 10 -5.0 1.5

X1 -0.09 -1.00 -0.82 -0.64 -0.09 1.00

X2 -1.00 0.14 0.14 0.14 0.14 0.14

X3 -1 -1 -1 -1 -1 -1

X4 1 1 1 1 1 1

Y: Pb Recovery (%) 47.0 40.2 49.6 56.0 72.4 77.4

1 2 3 4 5 6 7 8 9 10 11 12

5 60 15 5 60 5 60 60 5 20 55 20

16 2 10 16 16 16 2 16 2 4 10 10

-2.0 -3.5 -2.0 -5.0 -5.0 -3.5 -2.0 -2.0 -2.0 -5.0 -2.0 -3.5

1.5 0.5 1.0 1.0 0.5 0.5 1.5 1.0 0.5 0.5 0.5 1.5

-1.00 1.00 -0.64 -1.00 1.00 -1.00 1.00 1.00 -1.00 -0.45 0.82 -0.45

1.00 -1.00 0.14 1.00 1.00 1.00 -1.00 1.00 -1.00 -0.71 0.14 0.14

1 0 1 -1 -1 0 1 1 1 -1 1 0

1 -1 0 0 -1 -1 1 0 -1 -1 -1 1

68.8 22.0 24.6 31.2 29.6 25.8 34.0 47.2 5.0 19.4 12.4 18.0

IV bis 1 bis 5 bis

15 5 60

10 16 16

-5.0 -2.0 -5.0

1.5 1.5 0.5

-0.64 -1.00 1.00

0.14 1.00 1.00

-1 1 -1

1 1 -1

61.6 69.4 27.2

Table 5 – Determination of Pb content in Italian EVOO samples a

-1

C /ng g

EVOO sample

Pb

Proposed approach

b

MW digestion/ICP-QMS

A

1.18 (±0.12)

1.24 (±0.09)

B

0.63 (±0.10)

0.70 (±0.13)

C

2.80 (±0.15)

2.65 (±0.20)

a

Average values from 3 replicates, with standard deviation in parenthesis

b

Data calculated with the recovery correction of 72.5 ± 3.0%

Figure 1

-4

-1.8x10

-4

-2.0x10

-4

-2.2x10

-4

-2.4x10

-4

I (A)

-1.6x10

0

4

8

12

16

t (min)

Figure 2

20

24

28

32

Figure 3

Pb Recovery %

100

80

60

40

20

0 0

10

20

30

tdep (min)

Figure 4

40

50

60

Response Surface

a. 90

80

70

Response 60

50

40

tox

tdep

30

20

b. 95

85 90 0.5

80

75

0.0

65

tox

70

60

55

50 45

40

-0.5

35 30 25 -0.5

0.0

tdep

Figure 5

0.5

Highlights

•

The electrochemical preconcentration of Pb is performed directly in olive oil

•

A Pt working electrode is used as deposition/reoxidation device of the metal ions

•

D-Optimal Design is applied to optimise the experimental electrochemical parameters

•

After medium exchange, the Pb content is quantified by GFAAS or ICP-QMS

Declaration of interests X The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: