Total Mercury, Inorganic Mercury and Methyl Mercury Determination in ...

Food Anal. Methods (2012) 5:505–511 DOI 10.1007/s12161-011-9273-6

Total Mercury, Inorganic Mercury and Methyl Mercury Determination in Red Wine Valderi Luiz Dressler & Clarissa Marques Moreira Santos & Fabiane Goldschmidt Antes & Fabrina Regia Stum Bentlin & Dirce Pozebon & Erico Marlon Moraes Flores

Received: 8 May 2011 / Accepted: 6 July 2011 / Published online: 29 July 2011 # Springer Science+Business Media, LLC 2011

Abstract This study deals with the development of a method for total Hg, inorganic Hg and methylmercury (MeHg) determination in red wine by using flow injectioncold vapour generation–inductively coupled plasma mass spectrometry (FI-CVG-ICP-MS) and gas chromatographyICP-MS (GC-ICP-MS). For Hg speciation analysis, a derivatization step was carried out using a 1% (m/v) sodium tetraphenylborate (NaBPh4) solution, followed by extraction of Hg species and their quantification by GC-ICP-MS. The main parameters evaluated were the make-up gas flow rate, volume of the NaBPh4 solution, time for derivatization reaction/analyte extraction and solvent used for Hg species extraction. Accuracy was evaluated by analyte recovery, whereas recoveries ranged from 99% to 104% for Hg(II) and MeHg. The limits of detection (LODs) for Hg(II) and MeHg were 0.77 and 0.80 μg L−1, respectively. Wine from Argentina, Brazil, Chile and Uruguay were analysed. The wine samples were also acid digested for total Hg determination by FI-CVG-ICP-MS. The LOD of the method used for total Hg determination was 0.01 μg L−1. The concentrations of Hg species in red wine measured by GC-ICP-MS were lower than the respective LODs. Only total Hg was detected in the analysed samples, where the highest concentration of Hg found was 0.55±0.02 μg L−1. V. L. Dressler (*) : C. M. M. Santos : F. G. Antes : E. M. M. Flores Departamento de Química, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil e-mail: [email protected] F. R. S. Bentlin : D. Pozebon Instituto de Química, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, RS, Brazil

Keywords Mercury speciation . Red wine . FI-CVG-ICP-MS . GC-ICP-MS

Introduction Wine is one of the most traditional and consumed beverages worldwide. It has a complex chemical composition, including organic compounds such as sugars, ethanol, organic acids, tannins, aromatic and colouring substances, metals and metalloids (Minoia et al. 1994; Bentlin et al. 2011; World Health Organization 1990). Trace elements are also found in wine, as a consequence of elements uptaking from soil and groundwater by the grape vine. Trace elements can also be introduced during wine processing and storage and/or as a result of fertilizers and fungicides used in the vineyard or airborne particulate deposited on grapes in vineyard (Karadjova et al. 2004; Pohl 2007; Ibanez et al. 2008). The content of several elements that are important concerning to organoleptic (Cu, Fe, Mn and Zn) or toxicological (As, Cd, Hg and Pb) features has to be routinely monitored in wine. However, the toxicity of a given element is strongly dependent of the chemical form of the element. In the case of Hg, the organic species of the element are much more toxic than the inorganic species. The most important of them is methylmercury (MeHg) whose lethal dose ranges from 20 to 60 mg kg−1 for a 70-kg person (World Health Organization 1990). The higher toxicity of organic Hg species is attributed to their lypophilic properties and low elimination rate from the organism causing bioaccumulation (Mason and Benoit 2003). MeHg is efficiently absorbed in the gastrointestinal tract and then cross the blood–brain and placenta barriers, causing irreversible damages in the central nervous system (Leermakes et al. 2005).

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According to the International Organization of Vine and Wine (OIV), the maximum concentration of total Hg permissible in wine is 10 μg L−1 while cold vapouratomic fluorescence spectrometry is the technique recommended for Hg determination. However, despite the high toxicity of organic Hg, there is no recommendation regarding its determination in wine as well as the upper limit of organic Hg concentration allowable. The literature about Hg speciation in wine is scarce and most of the works found are focused on total Hg determination (Karadjova et al. 2004; Martinis and Wuilloud 2010). Mercury speciation is in general performed using chromatographic (Pilz et al. 2010; Grinberg et al. 2003; Rodrigues et al. 2010) or non-chromatographic (Kaercher et al. 2005; Duarte et al. 2009; Flores et al. 2009; Torres et al. 2009; Tuzen et al. 2009a, b) methods in conjuction with spectrometric techniques. Inductively couppled plasma mass spectrometry (ICP-MS) hyphenated with liquid chromatography (LC) or gas chromatography (GC) is very appropriate for Hg speciation analysis, in view of the high sensitivity, multielemental capability and good resolution (Dressler et al. 2011; Popp et al. 2010; Adams 2004). When LC is used, reversed-phase columns are commonly employed for separation of Hg species whereas organic solvent is used as mobile phase. The organic solvent content in the mobile phase must be as low as possible to avoid interferences such as plasma quenching and carbon deposits on the ICP-MS spectrometer interface. The effects of organic solvents in plasma source are described elsewhere (Dressler et al. 1998, 1999; Wangkarn and Pergantis 1999). If the LC separation is followed by chemical vapour generation (CVG) of Hg, the organic solvent can be separated. Then, only the volatile Hg species are introduced into the plasma that prevents interferences by organic solvent (Pilz et al. 2010; Krishna et al. 2010; Chiou et al. 2001). Speciation analysis of Hg using GC-ICP-MS requires a derivatization step to convert less volatile Hg species into more volatile species. The derivatization reaction carried out for Hg speciation analysis by GC-ICP-MS consists of ethylation with sodium tetraethylborate (NaBEt4) (Jackson et al. 2009) or propylation with sodium tetrapropylborate (NaBPr4) (Carrasco et al. 2009) or phenylation with sodium tetraphenylborate (NaBPh4) (Cai et al. 2000). It is worth citing that the addition of NaBEt4 would make impossible the determination of natural ethylated mercury species in the sample (Carrasco et al. 2009). Despite the derivatization step, speciation analysis of Hg by GC-ICP-MS is in general simpler and faster than by LC-ICP-MS. In addition, better limits of detection (LODs)s are expected in GC-ICP-MS (Jackson et al. 2009) because all injected sample is transported to the ICP. In LC-ICP-MS, the mobile phase leaving the separation column is introduced into the ICP by

Food Anal. Methods (2012) 5:505–511

using nebulisation where more than 95% of the mobile phase containing the analyte is discarded. Red wine is a complex matrix that cannot be directly analysed using LC-ICP-MS to prevent damage of the chromatographic column. Besides, the sample preparation for subsequent Hg speciation analysis requires mild conditions to avoid degradation or reaction of Hg species. In the case of GC-ICP-MS, the derivatized Hg species are extracted with an organic solvent that is then injected in the chromatograph. However, the volume of organic solvent injected is very low, which prevents plasma disturbance and interferences. The main objective of this study is to evaluate the applicability of GC-ICP-MS for inorganic Hg and MeHg speciation in red wine. Mercury species present in the wine samples are extracted with hexane after their reaction with NaBPh4 and then quantified using GCICP-MS. Parameters such as make-up gas flow rate, volume of NaBPh4 solution (used for derivatization), time for derivatization reaction/analyte extraction and type of solvent used for Hg species extraction are evaluated in order to achieve low LODs, good peak separation and shortest period of analysis as possible. Additionally, total Hg is determined by flow injection-cold vapor generationICP-MS (FI-CVG-ICP-MS).

Experimental Instrumentation A Hewlett Packard 5890 Series II gas chromatograph was used for Hg species separation. The chromatograph was connected to an inductively coupled plasma mass spectrometer (ELAN DRC II from PerkinElmer/Sciex, Canada), equipped with a quartz torch (injector tube with 2 mm, i.d.) and platinum cones. A homemade transfer line was used for the connection, being the heating controlled using the software of the GC equipment. Dry plasma conditions were used throughout the measurements by GC-ICP-MS. The WinFAAS 1.0 software was used for integration of the chromatographic signals (in peak area). The GC-ICP-MS operating conditions are described in Table 1. Total Hg was determined by FI-CVG-ICP-MS. A FICVG system built in house and described elsewhere (Kaercher et al. 2005) was fitted to the ICP-MS equipment. The FI-CVG system consists basically of a peristaltic pump (Gilson-Miniplus, France), a manual injector and a U-type gas–liquid separator. Tygon tubing was used to transport the solutions by the peristaltic pump: 1.14 mm i.d. for the reductant (0.1% m/v NaBH4) and sample, and 1.69 mm i.d. for the acid solution (1.0 mol L-1 HCl). Water


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Table 1 GC-ICP-MS operating conditions

1,300 15 1.10

dissolving the solid reagent in 0.1% (m/v) NaOH (from Merck). A stock solution containing 1,000 mg L−1 inorganic Hg (as Hg(II)) in 2% (v/v) HNO3 was purchased from Merck. A stock solution containing 1,000 mg L−1 of organic Hg was prepared by dissolving CH3HgCl (from SigmaAldrich) in methanol. Solutions with different concentration of Hg(II) and CH3HgCl were prepared by serial dilution of the stock solutions in 1.0 mol L−1 HCl. Calibration solutions were prepared fresh daily by serial dilution of the Hg(II) and CH3HgCl stock solutions in acetic acid/ sodium acetate buffer prior to derivatization. All Hg(II) and CH3HgCl solutions were stored in glass vessels, which were previously cleaned by immersion in 20% (v/v) HNO3 for 24 h and then rinsed with purified water. The flasks with the Hg(II) and CH3HgCl solutions where kept at 4 °C in the dark before use.

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Samples and Sample Preparation

GC GC inlet

On column

Column

PDMS (15 m×0.32 mm i.d.; 0.10 μm)

Injector temperature (°C) Volume injected (μL)

200 2

Carrier gas

Hydrogen

Carrier gas pressure (psi) Transfer line temperature (°C)

60 200

Oven temperature

50 °C min−1, 50 °C min−1 to 200 °C, program hold for 1 min

ICP-MS RF power (W) −1

Plasma gas flow rate (L min ) Make-up gas flow rate (L min−1) Dwell time (ms) Isotope monitored (m/z)

was used as sample carrier. Sample solutions were mixed on line with the HCl and NaBH4 solutions through a Ttype connector (0.8 mm i.d.). The mixture was pumped to the gas–liquid separator followed by Hg determination by ICP-MS. Argon (99.996%, from White Martins-Praxair, Brazil) was used as plasma gas and carrier gas. Hydrogen (99.999%, from White Martins-Praxair.) was used as mobile phase in gas chromatography. Reagents and Solutions All reagents used were of analytical grade. Purified water (18.2 MΩ cm, using a Milli-Q system from Millipore, Billerica, MA, USA) was used to prepare reagents and calibration solutions. Hydrochloric acid 37% (m/m) from Merck (Darmstadt, Germany) was used. Before using, this acid was purified by sub-boiling distillation (Duopur distiller from Milestone, Sorisole, Bergamo, Italy). Solutions containing 1% (m/v) NaBPh4 or 1% (m/v) NaBEt4 (both reagents from Sigma-Aldrich, St. Loius, MO, USA) were prepared in water and used for Hg derivatization. The pH of the calibration solution or sample was adjusted with 1.0 mol L−1 acetic acid/sodium acetate buffer (pH 4.90, prepared using acetic acid and sodium acetate, both reagents from Merck). The derivatized Hg species were extracted with 1.0 mL of hexane or toluene or isooctane (all from Mallinckrodt, St. Paul, MN, USA). Better results were obtained for hexane and for that reason only this reagent was used for Hg species extraction in wine. Sodium tetrahydroborate (NaBH4, from Vetec, RJ, Brazil) solution (0.1%, m/v) was prepared fresh daily by

Red wine produced in South America (in Argentina, Brazil, Chile and Uruguay) were analysed. Bottles (20) of red wine were purchased in local supermarkets. Samples were acid digested for total Hg determination. In this case, 10 mL of wine were transferred to glass flask (30 cm high×2 cm i.d.) to which 3 mL of HNO3 were added. Then, the flask was placed on a metallic block (from Velp Scientifica, Usmate, Milano, Italy) and heated at 60 °C for 2 h. After cooling at room temperature, the obtained solution was transferred to a graduated polypropylene vial and the volume completed to 20 mL by addition of water. This solution was used for total Hg determination by FI-CVG-ICP-MS. For Hg speciation analysis, 5 mL of wine or calibration solution mixed with 5 mL of wine were transferred to a glass vial to which 4 mL of acetic acid/sodium acetate buffer were added in order to obtain a pH of 4.90. Next, 1 mL of hexane and 0.5 mL of 1% (m/v) NaBPh4 were added to the vial, which was then capped and placed in an orbital water bath shaker. The mixture was shaken for a period of 30 min at room temperature for Hg species extraction into the organic phase. Then, the mixture was centrifuged at 3,000 rpm for 10 min to separate the aqueous and organic phases. The organic phase was transferred to a 2-mL autosampler vial (of amber colour) that was kept at −20 °C until analysis by GC-ICP-MS. The extracts were prepared in triplicate, whilst 2 μL of each triplicate were injected in the chromatograph. Standard addition calibration was used, where a calibration curve with five points was obtained. The concentration of Hg(II) and MeHg in the calibration solutions ranged from 1.00 to 25 μg L−1 and from 25 to 1,000 ng L−1 for total Hg determination. Since there is no certified red wine, accuracy was evaluated by analyte recovery tests.

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Results and Discussion Method Development Evaluation of Instrumental Conditions Firstly, chromatographic conditions were evaluated for obtaining good peak resolution in the shortest time possible. It was observed that by using a heating rate of 50 °C min−1, from 50 to 200 °C, separation and detection of Hg(II) and MeHg were possible within 5 min. The make-up gas was the nebulizer gas normally used in ICP-MS. The make-up gas transport the volatile Hg species from the GC column to the ICP. It is important to cite here that the transfer line must be fully heated, and the heating controlled, to avoid analyte condensation (Bouyssiere et al. 2002). The transfer line heating can be easily controlled for Hg speciation, but the make-up gas flow rate requires carefully optimization. In this work, the make-up gas flow rate was evaluated in the range of 1.00 to 1.40 Lmin−1, using a solution containing 25 μg L−1 of Hg(II) and MeHg. This solution was treated the same way as the samples and calibration solutions. The results obtained with respect to the make-up gas flow rate influence are shown in Fig. 1. The analyte signal (Fig. 1) is similar when the makeup gas flow rate is 1.00 and 1.10 Lmin−1. The analyte signal decreases for make-up gas flow rate higher than 1.10 Lmin−1. Therefore, it was fixed in 1.10 Lmin−1 for further measurements. Evaluation of Derivatization and Extraction of Hg Species in Red Wine According to the literature, the reagents NaBPr4 and NaBPh4 are mostly employed for Hg derivatization in Hg speciation analysis (Cai et al. 2000). In this study, NaBPh4

was used because it is cheaper than NaBPr4. An aliquot of 5 mL of red wine spiked with 25 μg L−1 of Hg and MeHg was placed in a glass vial and used to evaluate the derivatization procedure. This aliquot of wine was treated the same way as previously described for samples and calibration solution preparation, excepting the volume of the 1% (m/v) NaBPh4 solution. In this case, the volume of the 1% (m/v) NaBPh4 solution added ranged from 0.25 to 2 mL. The effect of NaBPh4 concentration is shown in Fig. 2. According to Fig. 2, at least 0.5 mL of a 1% (m/v) NaBPh4 (5 mg of the reagent) solution for quantitative MeHg derivatization is necessary. The signal intensity was practically the same for higher volumes of NaBPh4 solution added, demonstrating that 0.5 mL of NaBPh4 was enough for the derivatization of MeHg. The signal of Hg(II) is quite similar for all amounts of NaBPh4 added and even 0.25 mL of 1% (m/v) NaBPh4 solution was sufficient for Hg(II) derivatization. The lower signal of MeHg observed for lower amount of NaBPh4 is probably due to incomplete derivatization. The sample matrix can interfere because other compounds present in the sample also react with NaBPh4 or the sample matrix can hinder the derivatization reaction (Tang and Wang 2007). Because of these effects, an excess of the derivatizing reagent is recommended. The signal intensities of Hg(II) and MeHg shown in Fig. 2 are similar for 0.5 to 2.0 mL of 1% (m/v) NaBPh4 solution added to the red wine aliquot spiked with the Hg species. Keeping in mind that the concentrations of Hg(II) and MeHg are the same and ICP-MS response is element specific, it is possible to conclude that extraction efficiency using 1 mL of hexane was also similar for both species. Experiments were also carried out using toluene or isooctane to extract the derivatized Hg species. However, lower analyte signal intensity was observed, representing incomplete analyte extraction. Therefore, it was concluded 600 MeHg

600 MeHg

Hg

Hg

500

Intensity, counts

Intensity, counts

500 400 300 200

300 200 100

100 0

400

0 1.00

1.10

1.20

1.30

Make up gas flow rate, mL min-1

Fig. 1 Influence of the make-up gas flow rate on the signal intensity of Hg(II) and MeHg added to red wine and measured by GC-ICP-MS. Error bars represent the standard deviation of three measurements

0.25

0.5

1.0

2.0

1% (m/v) NaBPh4, mL

Fig. 2 Influence of the NaBPh4 amount used for derivatization of Hg (II) and MeHg added to red wine and measured by GC-ICP-MS. Error bars represent the standard deviation of three measurements


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that hexane was the most appropriate solvent for phenylated Hg species extraction. The time necessary for derivatization reaction and Hg species extraction was evaluated in the range of 5 to 40 min. An aliquot of 5 mL of red wine was spiked with 25 μg L−1 of Hg(II) and MeHg solution and treated as previously described for samples and calibration solution preparation, excluding the period of shaking. Figure 3 shows that better results were obtained when the mixture was shaken for 30 min, demonstrating more efficient derivatization/analyte extraction. Therefore, 30 min was chosen for derivatization and Hg species extraction.

Table 2 Total Hg concentration in red wine samples Sample

Origin

Grape variety

Total Hg (μg L−1)

1 2

Argentina Argentina

Syrah Pinot Noir