# Streamlining metal analysis in wine: Matrix purification for simple lead detection by AAS or portable electrochemical sensor

Taisiia Sikolenko, Elias Bou-Maroun, Laurence Noret, Marc Legal, Thomas Karbowiak

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Taisiia Sikolenko, Elias Bou-Maroun, Laurence Noret, Marc Legal, Thomas Karbowiak. Streamlining metal analysis in wine: Matrix purification for simple lead detection by AAS or portable electrochemical sensor. Food Chemistry, 2025, 491, pp.145047. 10.1016/j.foodchem.2025.145047. hal-05217950

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https://institut-agro-dijon.hal.science/hal-05217950v1

Submitted on 21 Aug 2025

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# Streamlining metal analysis in wine: Matrix purification for simple lead detection by AAS or portable electrochemical sensor

Taisiia Sikolenko<sup>a,b</sup>, Elias Bou-Maroun<sup>a</sup>, Laurence Noret<sup>a</sup>, Marc Legal<sup>b</sup>, Thomas Karbowiak<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Wine

Lead

Sample preparation

Solid phase purification

Graphite furnace atomic absorption

spectroscopy

Electrochemical sensor.

## ABSTRACT

This study presents a streamlined and gentle approach to the preparation of wine samples for lead determination, eliminating the need for concentrated acid digestion. The method relies on a pass-through solid-phase extraction (SPE) cleanup employing a polymeric styrene-divinylbenzene solid sorbent to selectively purify the wine matrix. Lead was quantified using either graphite furnace atomic absorption spectroscopy (AAS) or a portable electro-chemical sensor (MetalSens, OliSens, France). Ultraperformance liquid chromatography (UPLC) analysis of phenolic acids and flavonoids confirmed that the purification step effectively removes most organic interferences without compromising lead detection, with recovery rates ranging from 94 to 101 %. This proposed matrix cleanup minimizes fouling of the pyrolytic AAS platform, enables reliable reuse of SPE cartridges for up to five cycles, and facilitates rapid and accurate electrochemical lead determination in wine, with deviations within 15 % of reference AAS measurements.

## 1. Introduction

Wine is an alcoholic beverage of high social significance. According to the latest report of the International Organisation of Vine and Wine (OIV) as of 2022, world wine production is estimated at 258 million hectoliters, while its consumption is evaluated at 232 million hectoliters (International Organization of Vine and Wine, 2022).

From a chemical point of view, wine is a complex matrix based on a water-ethanol solution containing a number of organic (e.g., sugars, acids, proteins, tannins, phenolic and volatile organic compounds, etc.) and inorganic (namely minerals) compounds (Garrido & Borges, 2013; Maltman, 2013). In light of the presence of a rich variety of substances in wine, it is often associated with positive impact on health when consumed moderately (Guilford & Pezzuto, 2011). However, the contamination of wine by heavy metals, particularly lead, may compromise the beneficial features of this beverage leading to renal, reproductive, and nervous disorders (Wani et al., 2015). According to data from European Food Safety Authority, presented in the recent OIV review, wine is among the main contributors to the total dietary intake of lead in Europe (International Organisation of Vine and Wine, 2020). This is why, despite all the actions taken over the past years, the importance of lead monitoring in wine remains highly relevant.

The most common methods for detecting the presence of Pb in wine are atomic absorption and atomic emission spectroscopies (Aceto et al., 2002; Grindlay et al., 2011). Electrochemical lead determination in wine using anodic stripping voltammetry (ASV) techniques is another known solution (Antunović et al., 2021; Green et al., 1997; Illuminati et al., 2013; Maciel et al., 2019), although up till now it has not yet become the primary method for this purpose. Even though direct analysis of lead in wine is achievable for both spectroscopic (Aceto et al., 2002; Ajtony et al., 2008; Plotka-Wasylka et al., 2018) and electrochemical determination (Green et al., 1997; Maciel et al., 2019; Salvo et al., 2003), sample pre-treatment is still recommended due to the high complexity of the wine matrix and the interferents that it carries (Grindlay et al., 2011; Pohl, 2007).

Classically, the decomposition of organic fraction by dry or wet digestion (including UV- and microwave-assisted digestion) is applied to wine samples before metal analysis (Artimon et al., 2009; González et al., 2008; Illuminati et al., 2013; Kim, 2004). This ensures the complete elimination of organic components and permits to work with purer samples, leading to lower spectral interferences for spectroscopic techniques. In case of electrochemical analysis of lead by ASV, digestion is almost inevitable, as it prevents pollution of the working electrode by organic molecules present in wine (the reported exceptions include

<figure><img src="image_3.png" /></figure>

<footnote><sup>a</sup> Université Bourgogne Europe, Institut Agro, INRAE, UMR PAM, F-21000 Dijon, France</footnote>

<footnote><sup>b</sup> Olisens, F-69006 Lyon, France</footnote>

<footnote>* Corresponding author at: Université Bourgogne Europe, Institut Agro, INRAE, UMR PAM, F-21000 Dijon, France.</footnote>

<footnote>E-mail address: thomas.karbowiak@institut-agro.fr (T. Karbowiak).</footnote>
mercury-based working electrodes (Green et al., 1997; Maciel et al., 2019; Salvo et al., 2003) and modified working electrodes (Antunović et al., 2021)).

Nonetheless, digestion requires the use of relatively large volumes of concentrated nitric acid (up to 10 mL per sample (Galani-Nikolakaki et al., 2002; González et al., 2008)) either alone or in a mixture with H₂O₂, HClO₄, H₂SO₄ or V₂O₅ (Grindlay et al., 2011; Pohl, 2007). Apart from the evident harmfulness of these reagents, this procedure is quite time-consuming and thus has a low analysis throughput (time is estimated range from about half an hour (González et al., 2008) and 6 h (Illuminati et al., 2013), depending on conditions). It demands specific equipment (closed-vessel digestion equipment, microwave digestion systems, or dry digesters), qualified laboratory personnel, and, obviously, proper laboratory conditions for both sample preparation and for conventional analysis by atomic spectroscopy.

Another milestone in the development of wine sample preparation for lead determination came with the attempt to use polymeric cartridges for solid-phase extraction. Their application was reported in combination with atomic absorption spectroscopy detection, although it follows the objective of pre-concentration of lead. In particular, the polyurethane foam loaded with a 2-(2-benzothiazolylazo)-p-cresol solution demonstrated its capacity to bind lead and pre-concentrate it on the polymeric solid phase in the form of a complex (Lemos et al., 2002). A similar approach was used by Fernandes et al., who employed polyethylene to bind the Pb complex with ammonium diethyldithiophosphate (Fernandes et al., 2003). Yet the studied polymers were capable of extracting the bonded metal, these procedures still required preliminary sample digestion to remove the organic components in the wine.

In this article, we introduce a softer and more environmentally friendly sample preparation procedure that does not require digestion for the regular monitoring of lead in wine samples and can be applied not only in laboratory conditions, but also beyond them. The proposed approach is based on pass-through cleanup of wine sample using adsorption onto a polymer that is capable of eliminating the majority of organic interferants that can perturb both spectroscopic and electrochemical Pb measurements. Conventional graphite furnace atomic absorption spectroscopy (AAS) and the portable electrochemical sensor MetalSens were applied for lead quantification in purified wine samples.

## 2. Materials and methods

### 2.1. Chemicals

Standard solutions of Pb (1000 mg/L) in 2 % nitric acid and nitric acid (HNO₃, 68 %) were supplied by Carl Roth. NH₄H₂PO₄ phosphate modifier for graphite furnace (100 ± 2 g/L) and ultrapure water of LC-MS grade for AAS experiments were purchased from Merck. Methanol (≥99 %, Chem-Lab), acetonitrile (≥99.9 %, LC-MS grade, Honeywell), gallic acid (97.5–102.5 %, Sigma), Folin-Ciocalteu reagent (Chem-Lab), and sodium carbonate (≥99.5 %, anhydrous, Fisher Chemicals) were used. Sample preparation and electrochemical experiments were conducted using deionized water from Purelab.

### 2.2. Wine samples

Four wines were selected: two white wines, from the Aligoté and Chardonnay varieties (Aligoté and Bourgogne appellations, respectively), a rosé wine (Marsannay appellation), and a red wine from the Pinot Noir variety (Bourgogne appellation). All four wines were from the 2022 vintage and were supplied by the vineyard of the University of Burgundy. Wine samples were spiked with 100 µg/L of Pb shortly after

bottle opening and stirred for at least an hour for metal to integrate into the wine matrix.

### 2.3. Solid phase extraction for wine purification

#### 2.3.1. Preliminary tests

Prior to selecting the best-suited solid phase, potential candidates – namely Bond Elut ENV, Bond Elut Plexa, and Bond Elut PPL from Agilent; Oasis HLB cartridges from Waters; and CHROMABOND HR-X cartridges from Macherey-Nagel – were tested for their wine matrix purification capabilities. Their detailed characteristics are reported in supplementary information SI-Fig. 1. Visual observation for the red wine showed that the wine became clear after passing through all five solid phase extraction (SPE) cartridges, while the colored compounds stayed retained on the polymer phase. The intensity of sample coloration decreased in the following order: Oasis HLB > Bond Elut ENV > CHROMABOND HR-X > Bond Elut Plexa > Bond Elut PPL. The white wine samples became lighter in color after the SPE purification procedure as well, although, due to less intense initial yellowish coloration, the difference between the five purified samples was not visually noticeable. The analysis of polyphenolic content in the treated wines by ultra-performance liquid chromatography (UPLC) confirmed their lowest concentration in samples purified by the Bond Elut PPL cartridge, which drove the choice of this solid phase for the present work.

#### 2.3.2. Sample preparation protocol

The SPE procedure was carried out according to the protocol schematized in Fig. 1. Prior to introducing the wine sample, the Bond Elut PPL cartridges underwent conditioning with methanol (5.0 mL) and nitric acid at pH 3 (5.0 mL). The wine samples, after spiking with Pb (5.0 mL, 100 µg/L of Pb), were loaded onto the cartridge and were allowed to pass through it, after which they were collected. Rinsing of the cartridges was carried out with nitric acid at pH 3 (5.0 mL). This fraction was added to the filtered sample, resulting in 10 mL of purified wine sample, diluted two times. Loading and rinsing were conducted at an average flow rate of 1 mL/min. A control sample containing 100 µg/L Pb solution at pH 3.5 was prepared following the same procedure as for the wine samples.

For the reuse of SPE cartridges, they were washed following a sequence: methanol (5.0 mL), a solution of methanol and nitric acid (pH 1.5) in a 2:1 ratio (3.0 mL), a solution of acetonitrile and nitric acid (pH 1.5) in a 2:1 ratio (3.0 mL), and finally pure acetonitrile (2.0 mL), ending with final drying by purging air through the cartridge using a syringe. A total of 15 to 20 min per sample was required to complete the sample preparation sequence.

### 2.4. Pb quantification by graphite furnace atomic absorption spectroscopy

Lead concentration was analyzed using an Analytik Jena 600 AAS equipped with a prism and Neon correction. The intensity of the absorption was recorded at 283.306 nm. The temperature program was selected based on the recommendations provided by the Analytik Jena software Aspect LS for lead determination by AAS. It included three drying steps (80 °C and 90 °C, both for 20 s, and 110 °C for 10 s), pyrolysis (350 °C for 20 s and 800 °C for 10 s), atomization at 1500 °C (for 4 s) followed by cleaning at 2450 °C (for 4 s).

For graphite furnace atomic absorption spectroscopy Pb quantification, the Pb-spiked samples that did not undergo SPE treatment were diluted 4-fold with 1 % HNO₃, while the samples after matrix purification were diluted 2-fold with 1 % HNO₃ to match the calibration range. Calibration curves were obtained by injecting a 100 µg/L Pb solution (0, 2, 4, 6 µL), 0.52 M NH₄H₂PO₄ (2 µL) and 1 % HNO₃, to reach a total
<figure><img src="image_4.png" /><figcaption>Fig. 1. – Four-step solid phase extraction procedure for purification of wine samples prior to lead analysis by GF-AAS or electrochemical methods. (SPE: Solid Phase Extraction; MeOH: Methanol; HNO₃: Nitric Acid; ACN: Acetonitrile; Pb: Lead; GF-AAS: Graphite Furnace Atomic Absorption Spectrometry).</figcaption></figure>

volume of 20 µL (International Organisation of Vine and Wine, 2006). Measurements were carried out in triplicate.

## 2.5. Total polyphenol determination by the Folin-Ciocalteu method

Polyphenols were quantified through their oxidation with the Folin-Ciocalteu reagent, which consists of a mixture of phosphotungstic and phosphomolybdic acids. The analysis was conducted according to OIV recommendations by measuring absorbance using a UV-Vis spectrometer (Genesys 50 Thermoscientific) at 750 nm (International Organisation of Vine and Wine, 2009). For non-treated wine samples, 1.0 mL of white, 0.5 mL of rosé and 0.2 mL of red wine samples were respectively introduced into a 100 mL volumetric flask. Wine samples after SPE cartridge purification were diluted four times less than the untreated ones. The total content of polyphenols (in mg/L) was expressed in gallic acid (GA) equivalent (GA was taken as a representative polyphenolic compound), based on its calibration curve in the range of 0–10 mg/L.

## 2.6. Polyphenol identification and quantification by ultra-performance liquid chromatography

Before analysis by UPLC, wine samples were filtered using nylon filters (0.45 µm, Macherey-Nagel). Separation was performed on a Raptor ARD-18 column (1.8 µm, 150 × 2.1 mm) at 35 °C. The mobile phase composition, flow rate, and gradient were selected based on a previously optimized method (Popîrdă et al., 2022). Detection of wine polyphenols was carried out using a diode array detector at 525 nm (for anthocyanins), 320 nm (for caftaric, caffeic and coumaric acids, Grape

Reaction Product (GRP)), at 305 nm (for coumaric acid), at 280 nm (for GA, hydroxytyrosol, tyrosol, catechin, epicatechin, proanthocyanidin B1, proanthocyanidin B2, and dimer C), and at 260 nm (for protocatechuic and hydroxybenzoic acids) (Ballester et al., 2018; Popîrdă et al., 2022). Identification and quantification were performed using individual stock solutions (1000 ppm) prepared in methanol on the day of analysis, following the previously detailed procedure (Popîrdă et al., 2022). The total anthocyanin content was calculated as the sum of free anthocyanins with retention times below 45.20 min and for combined acylated and coumaroylated anthocyanins with retention times above 45.20 min.

## 2.7. Pb quantification by electrochemical analysis

Electrochemical detection of Pb was carried out using the portable electrochemical device MetalSens (OliSens, France), designed for heavy metal detection in drinking water and provided by OliSens company. The standard protocol for lead quantification in aqueous samples by anodic stripping voltammetry, based on the standard addition method, was applied (OliSens, 2023). The measuring conditions were set to an initial deposition of lead at -0.5 V for 15 s, followed by stripping in the potential range from E<sub>begin</sub> - 0.4 V to E<sub>end</sub> 0.0 V, with a E<sub>step</sub> of 0.006 V, amplitude of 0.02 V at a frequency of 75.0 Hz.

The described protocol was tested on two wine varieties - Chardonnay and Pinot Noir - after the 1st, 10th and 20th use of the SPE cartridge. A wine sample, after SPE purification (3.0 mL), was diluted in deionized water (22.0 mL) with the addition of an electrolyte solution (0.52 mol/L KCl and 1.3 mol/L HNO₃ in deionized water; 1.0 mL),
setting the target concentration of lead at 6 µg/L. Primary scanning of the diluted wine probe was followed by three standard additions of 100 µL of Pb at a concentration of 1 mg/L (measurements conducted in duplicates). All working solutions were kindly supplied by OliSens.

## 2.8. Statistical analysis

Statistical analysis was performed in Microsoft Excel using the XLSTAT add-in. A one-way analysis of variance (ANOVA) was used to compare the mean of lead concentrations at a 5 % level of significance. The limit of quantification for spectroscopic lead detection (LOQ<sub>AAS</sub>) was calculated based on the standard deviation of 14 blank measurements (SD<sub>noise</sub>) using eq. (1):

$$LOQ_{AAS} = 10 \times SD_{noise} \qquad (1)$$

This approach is recommended by the OIV for the evaluation of AAS method performance (International Organisation of Vine and Wine, 2006), and it provides a real-time, empirical assessment of the true detectability of the analyte, independent of curve fitting, minimizes potential variations of day-to-day calibration parameters, and reflects the actual performance of the used spectrometer.

For the electrochemical technique, the calculation of the limit of quantification (LOQ<sub>EC</sub>) was performed using the slope and the standard deviation of the intercept (SD<sub>intercept</sub>) of the standard addition curve, as presents eq. (2):

$$LOQ_{EC} = \frac{10 \times SD_{intercept}}{slope} \qquad (2)$$

The Cochran test was performed to evaluate whether the highest standard deviation within each sample group significantly differed from the others. The test statistic C for *m* number of measurement sets (*m* = 6) performed in triplicates (*n* = 3) was calculated following eq. (3) and was compared to C<sub>critical</sub>(α=0.05):

$$C = \frac{S_{max}^2}{\sum_{i} S_i^2} \qquad (3)$$

# 3. Results and discussions

## 3.1. Wine sample preparation by solid phase extraction

The proposed sample preparation procedure was tested on four types of wine – two white wines (from Aligoté and Chardonnay varieties), one red wine (Pinot Noir variety), and one Rosé wine (from Pinot Noir variety) – each with distinct organic composition and polyphenolic profiles. After solid phase extraction cleanup using Bond Elut PPL cartridge, it was observed that the majority of colored organic components were adsorbed onto the polymeric phase and stayed retained untill the washing procedure was performed. Rinsing the cartridge with 5.0 mL of nitric acid at pH 3 served two purposes: (i) to provide the complete transfer of the purified wine sample through the sorbent bed, and (ii) to release lead bound in organic complexes by converting it into free Pb<sup>2+</sup> ions, which are preferable for further electrochemical analysis. The collected fractions of 10 mL each containing Pb were typically transparent, uncolored, and visually homogeneous (SI-Fig. 2). A loss of sample coloration was already mentioned for the red wines after ultrafiltration passing from membranes with nominal molecular weight cut-off from 100,000 to 30,000, what was explained by a decrease of tannin concentration (McKinnon & Scollary, 1997).

The main objective behind using SPE sample preparation for wine in this study was to minimize the organic content of its matrix without altering the presence of Pb. Following this goal, a polymeric styrene-divinylbenzene solid phase was privileged over silica-based sorbents due to its stability over a wide pH range and its strong affinity for aromatic compounds, including polyphenols (De Villiers et al., 2004;

International Organisation of Vine and Wine, 2010). Moreover, it has already proven to be a suitable phase for the extraction of aromatic compounds, including polyphenols from wines (De Villiers et al., 2004; Domínguez et al., 2002; Jeffery et al., 2008; Liang et al., 2018; López et al., 2002; Matějíček et al., 2003). It is also expected that such polymeric phase remains inert or weakly reactive towards cations, allowing them to pass freely.

## 3.2. Pb quantification in wine by atomic absorption spectroscopy after solid phase extraction sample preparation

The results of Pb quantification before and after wine matrix purification are presented in Table 1. The proposed protocol for lead spectroscopic determination allowed to quantify Pb in white, rosé, and red wine samples with satisfactory recoveries. However, the internal camera revealed the formation of burnt residue on the pyrolytic platform following the analysis of each unpurified sample. After the application of the SPE cleanup protocol for wine preparation (Cycle 1 in Table 1), concentration of lead, as determined by AAS, did not significantly change compared to that in the unpurified wines. Recovery rates for Pb quantification varied from 96.3 % for red wine (Pinot Noir variety) to 102.0 % for white wine (Aligoté variety). In addition to the wine samples, a blank sample that contained 100 µg/L of Pb at pH 3.5 was subjected to the same sample preparation procedure. Similar to the wines, satisfactory recovery was found for lead in the blank sample (102.9 % for Cycle 1), indicating that the metal is not retained by the polymeric phase. The mean concentrations of lead obtained for the samples without sample preparation and those purified with the PPL cartridge did not show a statistically significant difference.

No effect of ethanol, the major organic compound present in wine, was noticed during Pb quantification when no matrix purification was performed, even though its interference was reported when quantifying metals in ethanol-containing solutions (Aceto et al., 2002). This may be due to the dilution applied, as well as the use of AAS with graphite furnace atomization over flame atomization, which is known to be less sensitive to minor variations in the surface tension of samples (Aceto et al., 2002). Additionally, pyrolysis of wine samples after solid phase purification allowed to avoid rapid pollution of the furnace caused by the decomposition of sugars and polyphenols (Ajtony et al., 2008; Artimon et al., 2009). Consequently, the platform was utilized for more than a hundred runs, while maintaining a stable background signal.

## 3.3. Impact of wine matrix purification on its chemical composition

The total polyphenol content in the starting wine samples showed an initial broad range of concentrations, increasing from white to red wines (Table 2), in accordance with previously reported data (Gutiérrez-Escobar et al., 2021; Markoski et al., 2016; Peña-Neira et al., 2000). The wine purification procedure conducted with Bond Elut PPL phase led to sufficiently lower concentrations of oxidizable matter. Particularly, its content decreased by 83 % in Aligoté, by more than 85 % in Chardonnay and Rosé samples, and by more than 95 % for Pinot Noir.

For better understanding which organic compounds were removed from the wine matrices, UPLC analysis was also conducted. The comparison of corresponding chromatograms of wine samples before and after matrix purification is presented in SI-Fig. 3. After Aligoté wine purification, none of the listed polyphenolic compounds was quantitatively identifiable (Table 2). For Chardonnay wine, apart from coeluted hydroxytyrosol and traces of GA that were detected, no other phenolic acids were found due to their low concentrations, even after a single use of the SPE cartridge. Traces of hydroxytyrosol and GRP were determined in the purified fraction of Rosé wine, likely as a result of the higher content of phenolic compounds compared to the white wines. Finally, for the red wine sample, Bond Elut PPL cartridge successfully removed the majority of tannins, anthocyanins, and polyphenols, except for residues of GA, caftaric acid and GRP.
<caption>**Table 1**</caption>

Concentration of Pb determined by AAS in various spiked wine samples and corresponding controls to evaluate the efficiency of SPE sample preparation and the reusability of the solid phase cartridge (up to 20 extraction cycles).

<table><thead><tr><th>Wine type</th><th>Number of successive SPE cycles performed</th><th>Mean C<sub>Pb</sub> ± Δ (P = 95 %, n = 3), (in µg/L)</th><th>SD C<sub>Pb</sub> (in µg/L)</th><th>RSD, (in %)</th><th>Recovery (in %)</th></tr></thead><tbody><tr><td rowspan="6">Aligoté<sup>A</sup></td><td>No sample preparation</td><td>102.0 ± 1.6<sup>a</sup></td><td>0.66</td><td>0.64</td><td>102.0</td></tr><tr><td>Cycle 1</td><td>101.7 ± 9.5<sup>a</sup></td><td>3.83</td><td>3.77</td><td>101.7</td></tr><tr><td>Cycle 5</td><td>102.6 ± 8.1<sup>a</sup></td><td>3.28</td><td>3.19</td><td>102.6</td></tr><tr><td>Cycle 10</td><td>99.9 ± 10.0<sup>a</sup></td><td>4.04</td><td>4.05</td><td>99.9</td></tr><tr><td>Cycle 15</td><td>101.4 ± 15.2<sup>a</sup></td><td>6.13</td><td>6.05</td><td>101.4</td></tr><tr><td>Cycle 20</td><td>93.7 ± 10.7<sup>a</sup></td><td>4.29</td><td>4.58</td><td>93.7</td></tr><tr><td rowspan="6">Chardonnay<sup>B</sup></td><td>No sample preparation</td><td>100.6 ± 8.0<sup>b</sup></td><td>3.22</td><td>3.21</td><td>100.6</td></tr><tr><td>Cycle 1</td><td>97.3 ± 13.4<sup>b</sup></td><td>5.39</td><td>5.54</td><td>97.3</td></tr><tr><td>Cycle 5</td><td>91.4 ± 11.9<sup>b</sup></td><td>4.77</td><td>5.22</td><td>91.4</td></tr><tr><td>Cycle 10</td><td>112.4 ± 6.1<sup>f</sup></td><td>2.44</td><td>2.17</td><td>112.4</td></tr><tr><td>Cycle 15</td><td>109.8 ± 10.3<sup>f</sup></td><td>4.13</td><td>3.76</td><td>109.8</td></tr><tr><td>Cycle 20</td><td>105.5 ± 8.6<sup>f</sup></td><td>3.45</td><td>3.27</td><td>105.5</td></tr><tr><td rowspan="6">Rosé<sup>C</sup></td><td>No sample preparation</td><td>101.5 ± 18.3<sup>c</sup></td><td>7.37</td><td>7.26</td><td>101.5</td></tr><tr><td>Cycle 1</td><td>96.3 ± 3.5<sup>c</sup></td><td>1.40</td><td>1.45</td><td>96.3</td></tr><tr><td>Cycle 5</td><td>100.7 ± 2.8<sup>c</sup></td><td>1.11</td><td>1.10</td><td>100.7</td></tr><tr><td>Cycle 10</td><td>100.8 ± 9.4<sup>c</sup></td><td>3.78</td><td>3.75</td><td>100.8</td></tr><tr><td>Cycle 15</td><td>103.1 ± 11.6<sup>c</sup></td><td>4.67</td><td>4.53</td><td>103.1</td></tr><tr><td>Cycle 20</td><td>94.7 ± 15.9<sup>c</sup></td><td>6.38</td><td>6.74</td><td>94.7</td></tr><tr><td rowspan="6">Pinot Noir<sup>D</sup></td><td>No sample preparation</td><td>99.5 ± 2.5<sup>C</sup></td><td>5.03<sup>C</sup></td><td>5.05<sup>C</sup></td><td></td></tr><tr><td>Cycle 1</td><td>96.4 ± 8.5<sup>d</sup></td><td>3.42</td><td>3.55</td><td>96.4</td></tr><tr><td>Cycle 5</td><td>96.3 ± 7.4<sup>d</sup></td><td>3.00</td><td>3.11</td><td>96.3</td></tr><tr><td>Cycle 10</td><td>101.8 ± 4.4<sup>d</sup></td><td>1.77</td><td>1.74</td><td>101.8</td></tr><tr><td>Cycle 15</td><td>103.4 ± 16.2<sup>d</sup></td><td>6.52</td><td>6.30</td><td>103.4</td></tr><tr><td>Cycle 20</td><td>101.7 ± 10.9<sup>d</sup></td><td>4.37</td><td>4.30</td><td>101.7</td></tr><tr><td rowspan="6">Blank<sup>E</sup></td><td>No sample preparation</td><td>100.1 ± 2.2<sup>D</sup></td><td>4.47<sup>D</sup></td><td>4.47<sup>D</sup></td><td></td></tr><tr><td>Cycle 1</td><td>100.5 ± 21.2<sup>e</sup></td><td>8.55</td><td>8.51</td><td>100.5</td></tr><tr><td>Cycle 5</td><td>102.9 ± 6.6<sup>e</sup></td><td>2.64</td><td>2.57</td><td>102.9</td></tr><tr><td>Cycle 10</td><td>98.7 ± 11.4<sup>e</sup></td><td>4.58</td><td>4.64</td><td>98.7</td></tr><tr><td>Cycle 15</td><td>101.8 ± 6.4<sup>e</sup></td><td>2.58</td><td>2.53</td><td>101.8</td></tr><tr><td>Cycle 20</td><td>101.1 ± 9.1<sup>e</sup></td><td>3.67</td><td>3.63</td><td>101.1</td></tr><tr><td rowspan="2"></td><td>No sample preparation</td><td>97.4 ± 8.3<sup>e</sup></td><td>3.33</td><td>3.41</td><td>97.4</td></tr><tr><td>Cycle 1</td><td>100.4 ± 2.2<sup>E</sup></td><td>4.38<sup>E</sup></td><td>4.37<sup>E</sup></td><td></td></tr></tbody></table>

n = 20 cycles of cartridge use; recovery – concentration of analyte found to that stated to be present, expressed in %, A-E – represent intraday statistical data for corresponding group, a-f – represent statistically equivalent groups.

Even though the elimination of polyphenols by the PPL Bond Elut phase has been successfully performed on various water samples, such as natural water (Masqué et al., 1998), seawater (Dittmar et al., 2008; Powers et al., 2019), treated wastewater (Gonsior et al., 2014), and drinking water (Munch, 2000), studies on wine are less common and have mostly focused on the determination of polyphenols in smoke-affected wines (Allen et al., 2013). The majority of results published on this topic are based on polyphenol extraction by OASIS HLB solid phase. Despite its proven efficiency for polyphenol pre-concentration from wine (Del Álamo et al., 2004; Liang et al., 2018; Manns & Mansfield, 2012), in the current study, the application of this phase was found to be the least performant. After preliminary tests, the red wine filtrate appeared to be the most heavily charged with polyphenols after OASIS HLB treatment compared to the other selected solid phases, meaning that the polymer did not provide the required wine purification. Nevertheless, it is possible that the introduced wine volume (5.0 mL) exceeds the capacity of OASIS cartridge (60 mg of polymer was used for 1.0 mL of wine sample (Manns & Mansfield, 2012)), compared to the Bond Elut PPL cartridge (1 g of polymer for 5.0 mL of wine sample).

The retention of wine polyphenols by the Bond Elut PPL phase is believed to be ensured by hydrophobic interactions and π-π stacking, as has been already shown for styrene-divinylbenzene polymers (Liang et al., 2018; Masqué et al., 1997). In parallel to surface interactions between the solid phase and organic compounds in wine, the latest also exist in equilibrium with lead, thus creating metal-organic complexes.

While dimeric polysaccharides such as rhamnogalacturonan-II (Szpunar et al., 1998), proteins and tannins (McKinnon & Scollary, 1997) are considered to be the dominant ligands that are capable of binding Pb in wines, a number of unidentified labile complexes can also be formed (Vasconcelos et al., 2000). It implies that there is a high chance for lead to be retained by the solid phase in its bound form. However, the results obtained in the present work demonstrate that Pb was quantitatively determined by AAS (Table 1). This can be explained either by the labile or dissociated form of lead present in the analyzed wine samples (Vasconcelos et al., 2000), or by the complexation of Pb with sizable molecules, such as dimers of pectic polysaccharides (Szpunar et al., 1998), proteins, and tannins (McKinnon & Scollary, 1997), which are not retained by the PPL phase. Despite the positive result presented, certain wines might still require preliminary acidification in order to ensure the release Pb from its complexes before the purification procedure (Aceto et al., 2002; Green & Scollary, 2000).

### 3.4. Reutilization of solid phase extraction cartridge

To investigate the possibility of using the Bond Elut PPL cartridge multiple times for the purification of wine matrix, they followed 20 successive cycles of conditioning, loading, and washing (following the procedure detailed in Fig. 1). One tested cartridge was reserved specifically for one analyte (wine sample or blank). It is worth mentioning that all samples collected from the first to the twentieth cycle appeared
<caption>**Table 2**</caption>

Quantification of total polyphenols in wine samples (by the Folin Ciocalteu method) before and after the first SPE purification of the sample and identification of selected organic compounds (by UPLC analysis) after the first and the twentieth use of SPE cartridge (referred as Cycle 1 and Cycle 20 respectively).

<table><thead><tr><th rowspan="2">Wine sample</th><th rowspan="2">Total polyphenol concentration<br>(in gallic acid equivalent, GAE in mg/L)</th><th colspan="4">Concentration of selected wine components identified after purification (in mg/L)</th></tr><tr><th>Before purification (mg/L)</th><th>After purification (mg/L)</th><th>Organic compound</th><th>Cycle 1</th><th>Cycle 20</th></tr></thead><tbody><tr><td rowspan="10">Aligoté</td><td rowspan="10">197.1 ± 3.6</td><td rowspan="10">33.1 ± 0.3</td><td>Hydroxytyrosol</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Tyrosol</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Gallic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Protocatechuic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Caftaric acid</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td>Hydroxybenzoic acid</td><td>&lt; 0.05</td><td>&lt; 0.05</td></tr><tr><td>Caffeic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Coutaric acid</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td>Coumaric acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>GRP</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td rowspan="10">Chardonnay</td><td rowspan="10">287.0 ± 4.8</td><td rowspan="10">35.0 ± 0.3</td><td>Hydroxytyrosol</td><td>Coelution</td><td>Coelution</td></tr><tr><td>Tyrosol</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Gallic acid</td><td>0.13</td><td>0.14</td></tr><tr><td>Protocatechuic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Caftaric acid</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td>Hydroxybenzoic acid</td><td>&lt; 0.05</td><td>&lt; 0.05</td></tr><tr><td>Caffeic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Coutaric acid</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td>Coumaric acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>GRP</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td rowspan="10">Rosé</td><td rowspan="10">324.7 ± 27.</td><td rowspan="10">46.7 ± 1.9</td><td>Hydroxytyrosol</td><td>0.63</td><td>&lt; 0.1</td></tr><tr><td>Tyrosol</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Gallic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Protocatechuic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Caftaric acid</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td>Hydroxybenzoic acid</td><td>&lt; 0.05</td><td>&lt; 0.05</td></tr><tr><td>Caffeic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Coutaric acid</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td>Coumaric acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>GRP</td><td>0.20</td><td>0.24</td></tr><tr><td>Proanthocyanidin B1</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>(+)-Catechin</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Proanthocyanidin B2</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>(–)-Epicatechin</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Dimer C1</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>FMA</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>CACA</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td rowspan="10">Pinot Noir</td><td rowspan="10">2381.3 ± 22.7</td><td rowspan="10">95.1 ± 1.4</td><td>Hydroxytyrosol</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Tyrosol</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Gallic acid</td><td>0.65</td><td>0.83</td></tr><tr><td>Protocatechuic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Caftaric acid</td><td>0.55</td><td>0.69</td></tr><tr><td>Hydroxybenzoic acid</td><td>&lt; 0.05</td><td>&lt; 0.05</td></tr><tr><td>Caffeic acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Coutaric acid</td><td>&lt; 0.15</td><td>&lt; 0.15</td></tr><tr><td>Coumaric acid</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>GRP</td><td>0.15</td><td>0.20</td></tr><tr><td>Proanthocyanidin B1</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>(+)-Catechin</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Proanthocyanidin B2</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>(–)-Epicatechin</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Dimer C1</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>FMA</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>CACA</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Piceid piceatannol</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr></tbody></table>

GAE – Gallic Acid Equivalent; GRP – Grape Reaction Product; Anthocyanins FMA – Free Mono glycosylated Anthocyanins; CACA – Combined Acylated and Coumaroylated Anthocyanins.

similar, with no noticeable coloration for any of the tested wine samples (see **SI-Fig. 2**). The procedure for cartridge washing was selected based on a previous study, where subsequent elution of wine organic fractions from styrene-divinylbenzene OASIS HLB cartridges was conducted using acidified methanol and acetonitrile solutions (Manns & Mansfield, 2012).

AAS analysis of the collected samples confirmed the presence of lead

in the purified wines after the 5th, 10th, 15th, and 20th cycles of cartridge use (Table 1). In the case of consecutive sample loading, Pb concentrations varied from 93.7 to 102.6 µg/L for Aligoté, from 91.4 to 112.4 µg/L for Chardonnay, from 94.7 to 103.1 µg/L for Rosé, and from 96.3 to 103.4 µg/L for Pinot Noir. Comparison of the in-batch standard deviations of the determined Pb concentrations in triplicates (calculated for the 1st to the 20th cycles, m = 6) confirms that the maximum
standard deviations for each batch are not significantly larger than the
rest variances obtained within each wine group (based on the Cochran
C-test).

The values of recovery for defined Pb concentrations in Aligoté, Rosé, and Pinot Noir wines varied from 93.7 to 103.4 %, making it possible to compare this parameter of purification methodology with the requirements for Pb quantification in wine by AAS as proposed by the OIV c. Indeed, according to the OIV criteria, recovery is acceptable within the range of 80–106 %. However, for Chardonnay, the recovery range was wider (from 91 to 112 %) because of the high $C_{Pb}$ obtained for the sample after the 10th cycle of cartridge reuse. Interestingly, the polyphenolic profiles of the samples after the 1st and 20th cycles of Bond Elut PPL cartridge use were not much different (SI-Fig. 3). No changes were observed in the composition of Aligoté and Chardonnay wines after the 1st and the 20th cycles for the components that were identified (Table 2). Meanwhile, some minor deviations in the determined concentrations of hydroxytyrosol and GRP were found for Rosé wine, and of GA, caftaric acid, and GRP for Pinot Noir wine.

The higher content of residual organic compounds after sample preparation for Pinot Noir wine may be related to the higher standard deviation value for the quantity of lead (Table 1). Namely, the content of GA, caftaric acid and GRP is increasing for the Pinot Noir sample after the 10th and 20th cycles of cartridge run (Table 2). Consequently, this may also apply to non-identified compounds that were not retained by

the cartridge. Lead concentrations obtained for Aligoté, Rosé, and Pinot
Noir wine samples before and after SPE sample preparation did not show
significant difference during cartridge use for 20 cycles (groups a, c and
d in Table 1). Conversely, Chardonnay wine samples after the purifica-
tion procedure were not considered statistically equivalent to the
starting wines (groups b and f in Table 1).

Thus, even though the determination of Pb after cartridge reuse up to
the 20th cycle is achievable, it is still recommended to avoid reusing
cartridges for more than 5 cycles due to potentially higher standard
deviations, which could start to appear.

3.5. *Pb quantification in wine samples by electrochemical analysis*

Before testing the purified wine samples, the diluted wine was subjected to direct electrochemical quantification of Pb by MetalSens device, following the provided protocol for its operation (OliSens, 2023). It was found that these conditions of lead analysis in non-treated wine samples were unsatisfactory, as the lead electrochemical signal was not recorded. This is most probably due the presence of high concentrations of interfering organic matter. Contrary, after application of the previously proposed protocol for wine purification using SPE, lead became detectable with this electrochemical technique (Fig. 2).

In Chardonnay wine, 87.6, 104.2 and 89.6 µg/L of Pb were detected
for the 1st, 10th and 20th cycles of cartridge run, respectively. As for

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Pb quantification by electrochemical analysis (using the MetalSens device, *n* = 2) in a) Chardonnay, b) Pinot Noir and c) Blank samples after matrix purification with a PPL cartridge reused for 1, 10 and 20 cycles.</figcaption></figure>

<figure><img src="image_7.png" /></figure>
Pinot Noir wine, a descending trend was observed, with 85.8, 79.3, and 68.5 µg/L of lead found in corresponding collected samples. The recovery for the blank sample tested in parallel was calculated at levels higher than 85 % for three samples, which is in accordance with the results obtained for Chardonnay. Pb quantification in the Chardonnay sample is consistent with the OIV regulations for AAS determination (International Organisation of Vine and Wine, 2006).

The comparison between AAS and electrochemical analysis (by MetalSens device) for lead quantification in Chardonnay wine shows a minor difference in determined Pb concentration, that varies from 9 to 12 % after the SPE cartridge cleanups. Pinot Noir seems to be a more challenging matrix, resulting in a larger gap between AAS and MetalSens techniques. Starting from a 15 % deviation for the 1st cycle of cartridge use, it progressively increased to 30 % for the 20th. Lower concentrations of Pb obtained for the red wine can be explained by the presence of more organic compounds in the purified samples compared to the white wines. In addition, the concentrations of GA, caftaric acid and GRP found in the red wine are increasing with the successive use of the Bond Elut cartridge (Table 2). The corresponding chromatograms of Pinot Noir after the 1st and 20th cycles of cartridge use also indicate that some of non-identified organic compounds were not retained by cartridge. It is thus highly possible that they can potentially affect the Pb quantification accuracy.

These results demonstrate the high potential of polymeric cartridges to remove the major interferents from wine, thereby enabling the electrochemical quantification of lead in case if the direct analysis is not possible. The adjustments to the sample preparation procedure and the parameters for Pb electrochemical detection are essential in order to achieve higher recoveries of metal concentrations. Yet, matrix purification using polymeric SPE cartridges is a solid alternative to digestion-based preparation that fulfills the demands of portability, ease of use, and compatibility with electrochemical techniques.

## 4. Conclusions

Lead detection in wine typically relies on previous digestion of the sample in order to remove excess of organic components, ensuring accurate analysis, minimizing matrix interference, and concentrating the analyte to lower the limit of detection. This work demonstrates an alternative procedure to this classical sample preparation, based on SPE, which removes most of the organic compounds from the wine matrix without interfering lead content. This method allows further detection by AAS or even by a portable electrochemical sensor (MetalSens device).

To support conclusions on the efficiency and utility of matrix purification for lead quantification by both tested methods, before and after cleanup procedure, multiple lines of evidence were considered. These include visual observations of the samples (supported by photographs, SI-Fig. 2), quantification of total polyphenol content using the Folin-Ciocalteu method, and detailed quantitative analysis of individual phenolic compounds by ultra-performance liquid chromatography. Furthermore, visual inspection of the pyrolytic platform during AAS analysis revealed fewer combustion residues and less frequent platform replacement in purified samples. Lastly, a significant matrix effect was observed in unpurified wine samples analyzed with the MetalSens device, where lead could not be detected, in contrast to successful quantification after purification.

The procedure was evaluated using four different types of wine, revealing a reduction in total polyphenol content by a factor of 6 to 25, depending on the wine variety, and achieving lead recovery rates ranging from 94 % to 101 %. It was proved that the Bond Elut PPL cartridge for wine purification can be reused, yielding recoveries ranging from 91.4 to 106.4 % over 5 cycles for white, rosé, and red wines.

The limitations of this preparation include the inevitable sample dilution, as well as the incomplete removal of organic matter compared to classical digestion methods. Nonetheless, the advantages of such a

purification procedure are evident, since it does not require the use of dangerous chemicals or specific equipment. It is also easy to perform, scalable, and applicable even in the non-laboratory conditions. Therefore, it holds strong potential for fast electrochemical Pb screening as well as for conventional AAS Pb detection, particularly when working with beverages containing lower organic loads.

### CRediT authorship contribution statement

**Taisiia Sikolenko:** Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. **Elias Bou-Maroun:** Writing – review & editing, Validation, Supervision, Investigation, Formal analysis, Data curation, Conceptualization. **Laurence Noret:** Writing – review & editing, Formal analysis. **Marc Legal:** Writing – review & editing, Validation, Supervision, Conceptualization. **Thomas Karbowiak:** Writing – review & editing, Validation, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sikolenko Taisiia reports that part of the financial support was provided by French National Research Agency and part by Olisens. Marc Legal is the director of Olisens, whose company also provided equipment. The other authors declare no competing interest.

### Acknowledgements

This work was supported by the Agence National de la recherche (ANR) through the France Relance program in collaboration with Olisens. The authors thank the DIVVA platform (Institut Agro Dijon, Université Bourgogne Europe) for access to various equipment (graphite furnace AAS, UV-vis spectrometer and UPLC). The authors are deeply grateful to Bohdan Oliinyk, Fleur Marie Saint Germain, Camille Delorme from Olisens and to Ali Assifaoui and Pierre Fouilloux from Institut Agro Dijon for their insightful discussions and contribution to improving the methodologies presented in this work.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2025.145047.

### Data availability

Data will be made available on request.

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