Fernanda
dos Santos Depoi
,
Fabrina R. S.
Bentlin
and
Dirce
Pozebon
*
Universidade Federal do Rio Grande do Sul, Instituto de Química, 91501- 970, Porto Alegre, RS, Brazil. E-mail: dircepoz@iq.ufrgs.br; Fax: + 55 33087304; Tel: + 55 51 33087215
First published on 16th December 2009
This paper describes the development of a method for the determination of mercury in honey. Analyte pre-concentration/matrix separation is carried out by cloud point extraction (CPE), while cold vapour-optical emission spectrometry (CV-ICP OES) is used to detect the analyte. A careful analytical work was carried out in order to evaluate precision and accuracy of the method. Mercury was complexed with ammonium diethyldithiophosphate (DDTP) and Triton X-114 was used as surfactant. Parameters such as type and concentration of acid (HCl or HNO3 + KCl), concentrations of complexant, surfactant and reductant (NaBH4), dilution of the surfactant-rich phase and the mass of honey were evaluated. Two procedures of sample preparation were investigated: sonication in presence of L-cysteine and acid decomposition in closed vessel. Sonication was very effective for extraction of inorganic mercury (Hg2+), but it was observed the sample should be decomposed to ensure the quantification of total mercury. Certified dog fish liver, analyte recovery and comparison with an independent technique (cold vapour-inductively coupled plasma mass spectrometry) were used to evaluate the method. Analyte recovery close to 100% was observed when up to 2.0 g of honey were subjected to sonication or up to 1.0 g of honey was decomposed with acid. The enrichment factor (EF) obtained was 13 and the limit of detection (LOD) was 2.2 ng g−1. The Hg concentration in the analyzed samples was lower than the LOD.
The honey is a mixture of sugars (27–44% fructose, 22–41% glucose, 2.7–16% maltose and 1.5–3.0% sucrose), proteins, amino acids (up to 0.09%), vitamins and other elements (Na, 0.03 to 0.05%; K, 0.04 to 0.12%; Ca, 0.15 to 0.03%; Mg, 0.05 to 0.08%; Fe, 0.0005 to 0.004% and trace elements, up to 0.01%).4,5 The honey matrix is relatively complex and may interfere with the determination of trace elements. Techniques such as inductively coupled plasma mass spectrometry (ICP-MS),3 inductively coupled plasma optical emission spectrometry (ICP OES),2,6 atomic absorption spectrometry with electrothermal atomization (ETAAS),7 instrumental neutron activation analysis (INAA)8 and differential pulse anodic stripping voltammetry (DPASV)5 have been used for the determination of trace elements in honey.
Among the toxic elements, mercury deserves special attention, especially for its ability to accumulate in various organisms9 and being transformed into organic mercury, usually in the form of methyl mercury (CH3Hg+). The concentration of Hg is very low in honey and usually not detected, excepting the product coming from very specific and contaminated areas.10,11
Few studies were found concerning the determination of Hg in honey. Due to the low concentration of this element and the complexity of the honey matrix, the separation of the matrix and analyte pre-concentration are recommended. The cloud point extraction (CPE) may be suitable for this purpose. In short, the CPE is based on the property that aqueous micellar solutions of nonionic surfactants become turbid when conditions such as temperature, salt and surfactant are modified.12 As a result, the aqueous micellar solution is separated into two isotropic phases: a surfactant-rich (with small volume) and a surfactant-poor (aqueous) with greater volume. One element, when complexed, can be extracted and included in the surfactant-rich phase, thus leading to a pre-concentration and separation of the element. The selection of suitable complexant, pH, ionic strength, surfactant type and concentration, temperature, equilibrium reaction and centrifugation time are parameters that have to be examined to make CPE successful.
Different compounds have been used as complexant of Hg involving CPE: (5-bromo-2-pyridylazo)-5-diethylaminophenol [(5-Br-PADAP)]13 for Hg determination in biological samples by ETAAS; 5-Br-PADAP for Hg determination in water by ICP-OES;14 I-methylene blue and pyrrolidine dithiocarbamate (APDC) for Hg determination in seafood by ICP OES;15 and ammonium diethyl dithiophosphate (DDTP) for Hg determination in biological materials by cold vapour generation coupled to atomic absorption spectrometry (CV-AAS).16 DDTP is a very suitable complexing agent because it is stable in acidic medium.17–20 Furthermore, this reagent is sufficiently hydrophobic to be used in CPE. With respect to the surfactant, which is necessary for the formation of micelles, octylphenoxypolyethoxyethanol (Triton X-114®, a nonionic surfactant) has been more widely used,15,16,21 mainly because of the low cloud point temperature (between 22 and 25 °C) and low cost. It is noteworthy that the CPE has been applied to different types of matrix, but not for honey.
The main objective of this research is the development of a method for the determination of Hg in honey. To do this, Hg is pre-concentrated by CPE and then detected by CV-ICP OES (cold vapour generation-inductively coupled plasma optical emission spectrometry). DDPT is used as complexant, while Triton X-114® is used as surfactant. The main objectives include: (i) to obtain the largest enrichment factor as possible, (ii) a viable procedure of sample preparation, and (iii) low reagent consumption and low waste production.
Plasma power/W | 1500 |
Plasma gas flow rate/mL min−1 | 15 |
Auxiliary gas flow rate/mL min−1 | 0.2 |
Plasma view | axial |
Injector tube | alumina, 2 cm i.d. |
Resolution | High |
Spray Chamber | Cyclonic |
Nebulizer gas/L min−1 | 0.75 |
Nebulizer | GemCone (the sample uptake rate was 1 mL min−1) |
Integration time/s | automatic, 2.5–5.0 |
Spectral line/nm | 253.652 |
Replicates | 2 |
Background correction | 2 points per peak |
Signal processing | peak area (7 points per peak) |
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Fig. 1 Diagram of the system used for cold vapor generation. Ar: carrier gas (argon at a flow rate of 0.6 mL min−1), G/L: gas/liquid separator; W: waste; HCl and NaBH4 solutions concentration: 0.1 mol L−1 and 0.1% (m/v), respectively. PTFE (0.8 mm i.d.), orange-white Tygon (0.95 mm i.d., for NaBH4 and sample solutions) and red-red Tygon (4.5 mm i.d. for the HCl solution) tubing were used for the propulsion of the solutions. The peristaltic pump of the ICP OES spectrometer was used. |
For Hg determination by CV-ICP-MS (cold vapour-inductively coupled plasma mass spectrometry), an ELAN DRC II instrument (from PerkinElmer/SCIEX) was employed. For cold vapour generation, the system depicted in Fig. 1 was coupled to the instrument and used. In this case, the carrier gas was 1.2 L min−1. An ultrasonic processor (Unique, Brazil) equipped with a 4 mm diameter-titanium tip, and a heating block (TE-015/1 from Tecnal, Brazil) were used for the homogenization and/or extraction of Hg in honey. A water bath with temperature control (DeLeo, Brazil) was used as a source of heating and assists the CPE, whereas a centrifuge (Fanen, Baby® 206, Brazil) was used for the separation of phases.
Solutions of Hg2+ were prepared by appropriate dilution of a 1000 mg L−1 stock solution of the species (Titrisol®, Merck). Methyl mercury chloride (CH3HgCl) from Aldrich, containing 1000 mg L−1 of Hg in methanol was used. Intermediate solutions containing 1.0 mg L−1 of Hg in the form of Hg2+ or 1.3 mg L−1 of Hg in the form of CH3HgCl, both in 1.0% (m/v) HNO3 were prepared just before the calibration procedure. The Hg concentration in the calibration solutions ranged from 0.3 to 10 μg L−1 for determination using CPE and CV ICP OES, 1.0 to 10 μg L−1 for CV-ICP OES (without Hg pre-concentration), and from 10 to 30 μg L−1 for direct (pneumatic nebulization) determination using ICP OES.
Samples of bee honey from different sites in the South of Brazil and of different floral origin (eucalyptus, orange, Pluchea sagittalis and field flowers) were analyzed in this work. The reference material DOLT-3 (Dogfish Liver) from the National Research Council of Canada was analyzed for evaluating the accuracy and precision of the method.
With respect to the ligand (DDTP) concentration, the highest Hg signal was observed when the DDTP concentration was 0.05% (m/v) for both media (Fig. 2). This concentration is ten times lower than that reported in the literature,16 for pre-concentration of Hg based on CPE and determination by atomic absorption spectrometry with cold vapor generation (CV AAS). It is observed in Fig. 2 the analyte signal decreases with the DDTP concentration increasing. This may be due to the formation of charged species that can interact with the surfactant, and/or DDTP free molecules can interact with the surfactant and thus compete with the formation of DDTP-Hg molecules.25 With regard to the surfactant concentration, the highest analyte signal was observed for 0.3% (m/v) Triton X-114 in presence of HNO3 and 0.1% (m/v) Triton X-114 in presence HCl (see Fig. S3 in supplementary file).† However, if the surfactant concentration was 0.1% (m/v), the volume of the resulting surfactant-rich phase was very small and more difficult to see, making phases separation difficult. Thus, as a compromise condition, the concentration of Triton X-114 was fixed in 0.3% (m/v). It was observed that the signal of Hg decreases with the surfactant concentration increasing, and this was due to the increased volume of surfactant-rich phase, which increased dilution. It is important to mention that, regardless of the surfactant-rich phase volume, 100 μL methanol + 1000 μL of acid solution were added to it. The decrease of analyte signal with increasing concentration of surfactant had also been observed in other studies.
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Fig. 2 Effect of the ligand (DDTP) concentration on Hg (3.0 μg L−1 of Hg2+) pre-concentration. Pre-concentration medium: 0.5 mol L−1 HCl or 0.5 mol L−1 HNO3 + KCl 0.007% (m/v), and Triton X-114 0.3% (m/v). |
The reaction between Hg and NaBH4 is accelerated when in acidic medium. Therefore, the concentration of HCl aspirated and transported through the CV system (Fig. 1) to assist the Hg2+ reduction was also evaluated. It can be seen in Fig. 3 the signal of Hg decreases with the HCl concentration increasing, because of the excess of H2 produced, destabilizing the system and undermining the separation of mercury (Hg0). This effect is more pronounced when the extraction of Hg is performed in medium containing HCl. According to Fig. 4, the signal of Hg increases with the NaBH4 concentration increasing (up to 0.1% m/v), and then decreases. It was observed the reaction medium became turbulent in the presence of higher concentrations of NaBH4, which can influence the kinetics of the Hg2+ reaction reduction and separation of the mercury vapour generated. Thus, the concentration of NaBH4 was set in 0.1% (m/v) for further measurements.
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Fig. 3 Effect of the HCl concentration transported trough the CV system. A solution containing 3.0 μg L−1 of Hg2+ was used. Pre-concentration medium: 0.5 mol L−1 HCl or 0.5 mol L−1 HNO3 + KCl 0.007% (m/v), DDTP 0.05% (m/v), and Triton X-114 0.3% (m/v). |
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Fig. 4 Effect of the reductant concentration (NaBH4) on the Hg signal (3.0 μg L−1 of Hg2+ was used). Pre-concentration medium: 0.5 mol L−1 HCl or 0.5 mol L−1 HNO3 + KCl 0.007% (m/v), DDTP 0.05% (m/v), and Triton X-114 0.3% (m/v). |
Certified or spiked/μg g−1 | Found/μg g−1 | ||||
---|---|---|---|---|---|
Procedure | Sample | Hg2+ | CH3Hg+ | Hg2+ | CH3Hg+ |
Sonication | DOLT - 3 | 1.78 | 1.59 ± 0.12 | 1.74 ± 0.02 | undetected |
Honey | 0.250 | 0.125 | 0.251 ± 0.012 | 0.055 ± 0.012 | |
Hg Total | Hg total | ||||
Acid digestion | DOLT-3 | 3.37 ± 0.14 | 3.07 ± 0.50 | ||
Honey | 0.250 | 0.249 ± 0.007 |
The effect of the amount of honey was investigated for both procedures of sample preparation. The maximum mass of sample tested was 2.0 g because of the difficulty of decomposing larger honey mass in closed vessel. According to Fig. 5 (B), the recovery of Hg is about 100% for sample mass up to 1.00 g, when the honey is digested and the analyte pre-concentrated. However, the recovery is less than 85% (see A in Fig. 5) if the analyte is measured directly in the sample solution by CV-ICP OES. When the honey sample is sonicated in the presence of L-cysteine (see C and D in Fig. 5), recovery is almost 100% for the full range of sample mass, regardless of the separation of the sample matrix. It is believed that sonication does not destroy the sugars, which masks the interferents. According to the results achieved so far, the decomposition of the sample with subsequent addition of L-cysteine is the most appropriate, given the good recovery of the analyte and the possibility to determine the total Hg. On the other hand, if sonication is used in the presence of L-cysteine, a larger amount of honey sample can be used, improving the LOD. There are no published data in relation to organic mercury in honey, but it must be very low and not detected by the techniques currently available for routine work. Thus, the sonication procedure can be recommended for Hg extraction from honey.
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Fig. 5 Effect of amount of honey mass on Hg recovery. A: Decomposition with acid and direct determination; B: decomposition with acid and CPE; C: sonication and direct determination; D: sonication and CPE. The honey sample was spiked with Hg2+ in order to obtain 10 μg L−1 of the element in the sample solution. |
Parameter investigated | Selected conditions |
---|---|
Sample mass/g | 2.0 (sonication) 1.0 (acid digestion) |
DDTP concentration/% (m/v) | 0.05 |
L-cystein/% (m/v) | 0.2 |
Triton X-114/% (m/v) | 0.3 |
Reduction medium HCl/mol L−1 | 0.1 |
Volume of methanol added to the surfactant-rich, phase/μL | 100 |
Botanical origin | Found/ng g−1 | CV-ICP-MS/ng g−1 | Spiked sample/μg g−1 | Found in the spiked sample/μg g−1 | Recovery/% |
---|---|---|---|---|---|
a = found using CPE and CV-ICP OES; b : LOD of the developed method. | |||||
Eucalyptus | <2.2b | 1.67 ± 0.61 | 0.125 | 0.126 ± 0.018 | 104 |
Eucalyptus | 0.250 | 0.253 ± 0.015 | 105 | ||
Orange | <2.2b | 1.86 ± 0.39 | 0.125 | 0.120 ± 0.024 | 101 |
Orange | 0.250 | 0.242 ± 0.029 | 103 | ||
Pluchea sagittalis | <2.2b | 1.67 ± 0.90 | 0.125 | 0.123 ± 0.014 | 100 |
Pluchea sagittalis | 0.250 | 0.244 ± 0.020 | 100 | ||
Field Flowers | <2.2b | 1.44 ± 0.75 | 0.125 | 0.123 ± 0.004 | 102 |
Field Flowers | 0.250 | 0.238 ± 0.058 | 99.5 |
Footnote |
† Electronic supplementary information (ESI) available: Fig. S1: Scheme of the sample preparation and CPE procedures. Fig. S2: Influence of the acid medium on the pre-concentration of Hg. Fig. S3: Effect of the surfactant on the pre-concentration of Hg. Fig. S4: Effect of the volume of methanol added to the surfactant-rich phase. See DOI: 10.1039/b9ay00189a |
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