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Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure
Talanta 80 (2010) 1158–1163
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Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure Jairo L. Rodrigues, Samuel S. de Souza, Vanessa C. de Oliveira Souza, Fernando Barbosa Jr. ∗ Laboratório de Toxicologia e Essencialidade de Metais, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
a r t i c l e
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Article history: Received 16 July 2009 Received in revised form 31 August 2009 Accepted 1 September 2009 Available online 9 September 2009 Keywords: ICP-MS Speciation Blood samples Mercury Sample preparation Methylmercury Thimerosal Liquid chromatography
a b s t r a c t Despite the necessity to differentiate chemical species of mercury in clinical specimens, there are a limited number of methods for this purpose. Then, this paper describes a simple method for the determination of methylmercury and inorganic mercury in blood by using liquid chromatography with inductively coupled mass spectrometry (LC–ICP-MS) and a fast sample preparation procedure. Prior to analysis, blood (250 L) is accurately weighed into 15-mL conical tubes. Then, an extractant solution containing mercaptoethanol, l-cysteine and HCl was added to the samples following sonication for 15 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 5 min on a C18 reverse-phase column with a mobile phase containing 0.05% (v/v) mercaptoethanol, 0.4% (m/v) l-cysteine, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol. The method detection limits were found to be 0.25 g L−1 and 0.1 g L−1 for inorganic mercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). The proposed method was also applied to the speciation of mercury in blood samples collected from fish-eating communities and from rats exposed to thimerosal. With the proposed method there is a considerable reduction of the time of sample preparation prior to speciation of Hg by LC–ICP-MS. Finally, after the application of the proposed method, we demonstrated an interesting in vivo ethylmercury conversion to inorganic mercury. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Speciation analysis of clinical samples is gradually becoming more widely accepted for nutritional and/or toxicological purposes. According to International Union of Pure and Applied Chemistry (IUPAC), speciation analysis is defined as the analytical process of identifying and/or measuring quantities of one or more individual chemical forms in a sample, and speciation of an element is defined as the distribution of an element among defined chemical species in a system [1,2]. Mercury (Hg) is one of the most hazardous pollutants in the environment. It exists in three basic forms: elemental mercury (Hg0 ) known as metallic mercury, inorganic mercury compounds (Ino-Hg), primarily mercuric chloride, and organic mercury, primarily methylmercury (Met-Hg) [3,4]. Organic forms are more toxic than inorganic [4]. The main sources of human exposure to organic mercurials, mainly in the form of methylmercury (Met-Hg) or ethylmercury
∗ Corresponding author. Tel.: +55 16 36024701. E-mail address: [email protected] (F. Barbosa Jr.). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.09.001
(Et-Hg), are the consumption of fish [4] and vaccines containing thimerosal, respectively [5]. On the other hand, the most common forms of exposure to inorganic forms are by inhalation of Hg vapor released from dental amalgams or from gold mining activities [4]. Measurements of Hg in both blood and hair are used to determine whether adverse health effects are likely to occur. The contents of mercury species in blood may represent a cumulative exposure from the daily diet and/or occupational environment exposure. However, despite the difference in toxicity related to the form of mercury, total mercury concentration (THg) in blood is usually used to estimate the clinical outcomes after mercury exposure. Moreover, the proportion between chemical forms of mercury in blood may vary among individuals. This makes it essential to have analytical methods, based on speciation analysis, which can differentiate between chemical forms in blood to diagnose risks of toxicity. The most effective instrumental-based techniques for chemical speciation analysis rely on the use of chromatography (mainly gas chromatography (GC) [6–9] or liquid chromatography (LC)) [10–12] coupled to a specific and sensitive detector, such as inductively coupled plasma mass spectrometry (ICP-MS). Compared with GC, LC is
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the preferred separation technique used for mercury speciation, because the mercury species do not need to be derived to volatile compounds before HPLC separation. Although several methods have been developed for measuring mercury in blood samples [13–17], there is merely a few proposing speciation analysis [18,19]. Moreover, all of them suggested very tedious and time-consuming sample preparation procedures. Moreover, artifact formation during the sample preparation step is a potential source of error [20,21]. On the other hand, clinical laboratories must cope with an increasing demand for trace element analysis in body fluids and tissues in response to increasing concern for occupational and environmental exposure to mercury. Thus, fast sample preparation procedures with minimal handling are extremely desirable in routine analysis. The aim of this paper was therefore to evaluate a simple method for methylmercury and inorganic mercury determination in blood by high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) with a fast sample preparation procedure prior to analysis. The method was then applied for speciation of mercury in blood samples collected from a Brazilian riparians, living in the Amazon region, exposed to mercury from fish consumption. It was also demonstrated for the first time that ethylmercury is in vivo converted to inorganic mercury, making the determination of the inorganic form of mercury in blood relevant for thimerosal exposed populations.
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Table 1 Liquid chromatography and ICP-MS operating conditions for Hg speciation in blood samples. LC conditions Column Pre-column (Guard column)
Mobile phase flow rate Sample loop Measurement
C18 (5 m, 150 mm × 4 mm) RP18 (7 m, 15 mm × 3.2 mm) 0.05% (v/v) mercaptoethanol 0.4% (m/v) l-cysteine 0.06 mol L−1 ammonium acetate 5% (v/v) methanol 1 mL min−1 100 L Peak high
ICP-MS experimental conditions Radio frequency power (W) Scan mode Nebulizer gas flow (L min−1 ) Resolution (amu) Replicates Isotopes
1200 Peak hopping 0.58 0.7 3 202 Hg
Mobile phase
2.4. Reagents
A PerkinElmer model L-200 LC pump, six-port injector (Rheodyne 9725) with a reverse-phase column (C18, 5 m, 150 mm × 4 mm, Brownlee Columns PerkinElmer, USA) and a precolumn (guard column) RP18 (7 m, 15 mm × 3.2 mm, Brownlee Columns PerkinElmer, USA) comprised the LC system. Samples were loaded with a syringe into a 100-L sample loop. All separations were performed at room temperature under isocratic conditions. The isocratic mobile phase was 0.05% (v/v) mercaptoethanol, 0.4% (m/v) l-cysteine, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol. The flow rate was 1.0 mL min−1 . The effluent from the LC column was directly connected to the nebulizer with PEEK tubing (1.59 mm o.d.) and a low dead volume PEEK connector. Data evaluation was performed using Chromera® software supplied with the instrument, and quantification was based on peak high by external calibration. The optimum experimental conditions for both ICP-MS and LC are given in Table 1.
All reagents used were of analytical grade and the solutions were prepared using high-purity water with a resistivity of 18.2 M cm, obtained from a Milli-Q Plus water purification system (Millipore, Bedford, MA, USA). Nitric acid (Merck, Darmstadt, Germany), was doubly distilled in a quartz sub-boiling apparatus (Kürner Analysentechnik, Rosenheim, Germany). A clean laboratory and laminar-flow hood capable of producing class 100 were used for preparing solutions and samples. All solutions were stored in high-density polyethylene bottles. Plastic bottles and glassware materials were cleaned by soaking in 10% (v/v) HNO3 for 24 h, rinsed five times with Milli-Q water and dried in a class 100 laminar-flow hood before use. All operations were performed on a clean bench. A 10-mg L−1 standard solution of inorganic mercury was obtained from PerkinElmer (PerkinElmer, Norwalk, CT). A 1000mg L−1 standard solution of methylmercury chloride (CH3 HgCl) and 1000-mg L−1 standard solution of ethylmercury chloride (CH3 CH2 HgCl) in water were obtained from Alfa Aesar. Analytical calibration standards of mercury species were prepared daily over the range of 0.0–20.0 gL−1 for the LC–ICP-MS method by suitable serial dilutions of the stock solution in the mobile phase. Additional chemicals for the speciation studies were HPLC grade methanol (99.9%, v/v) and mercaptoethanol (Sigma–Aldrich, USA), l-cysteine (Fluka, Japan). Ammonium acetate (99.99%) was obtained from Aldrich Chemical Company (Milwaukee, USA). For the ICP-MS method for total mercury determination, Rh and Au were each diluted from 1000 mg L−1 stock standards (Assurance grade; Spex Certiprep® ). Triton® X-100 was obtained from the Sigma–Aldrich Co. (Sigma Ultra Grade, Sigma–Aldrich Co., St. Louis, MO, USA) and was diluted to 10% (v/v) with double-deionized water. A rinse solution consisting of 0.005% (v/v) Triton X-100® and 1 mg L−1 Au in 2% (v/v) nitric acid was prepared before each run.
2.3. Measurements of total mercury in blood
2.5. Sample preparation for speciation analysis
The method used for the determination of total mercury in blood samples was based on a previous publication described by Palmer et al. [13]. Briefly, blood specimens were diluted 1 + 49 into a 15mL polypropylene Falcon® tube (Blue MaxTM Jr., Becton Dickinson) with a diluent solution containing 0.5% (v/v) double-distilled HNO3 , 25 g L−1 Rh as internal standard, 1 mg L−1 Au to control Hg memory effects in the spray-chamber, and 0.005% (v/v) Triton® X-100 and analyzed by ICP-MS.
Blood samples (250 L) were placed in 15 mL polypropylene test tubes with 4.75 mL of a solution containing 0.10% (v/v) HCl + 0.05% (m/v) l-cysteine + 0.10% (v/v) 2-mercaptoethanol and then sonicated for 15 min in an ultrasonic bath 1400 A (UNIQUE, Brazil). The resulting solution was centrifuged and then filtered through 0.20 m Nylon® filters (Millipore, USA). Sample extraction was performed in triplicate and extraction blanks were prepared in the same manner.
2. Material and methods 2.1. Instruments and apparatus All measurements were made with an ICP-MS (Elan DRC II PerkinElmer, Norwalk, CT) for total mercury determination and for speciation. 2.2. Measuring of mercury species
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2.6. Standard reference materials and ordinary blood specimens
3.2. Evaluation of the time of ultrasound extraction
Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, MD). For additional validation purposes, human blood specimens (n = 10) collected as part of another research study involving Hg exposure in the Brazilian Amazon region were available for method comparison purposes with identifiers removed [22,23]. These specimens had been obtained with informed consent from human subjects in accordance with procedures approved by our Institutional Review Board.
For this experiment, it has been evaluated the extractor solution containing 0.10% (v/v) HCl + 0.05% (m/v) l-cysteine + 0.10% (v/v) 2mercaptoethanol and different times of ultrasound energy (from 0 to 30 min). Different aliquots (250 L) of the SRM NIST 966 were used for the experiment. Quantitative extraction of Hg (>95%) was observed for the NIST 966 blood by using the proposed extraction solution and 15 min of ultrasound energy. Then, for the subsequent experiments, mercury species were extracted from the blood samples by using the solution containing 0.10% (v/v) HCl + 0.05% (m/v) l-cysteine + 0.10% (v/v) 2-mercaptoethanol and 15 min of ultrasound energy.
2.7. Blood specimen collection Venous blood was collected into pre-screened, 3-mL draw lavender-capped BD Vacutainer® blood collection tubes containing K2EDTA anticoagulant (Becton Dickinson, Franklin Lakes, NJ, USA; part no. 367856). These tubes were used with BD Vacutainer® EclipseTM 22 gauge, 1–1.25 in. blood collection needles (Becton Dickinson; part no. 368608). After blood was drawn into the Vacutainer® , the tube was inverted several times to mix blood with K2EDTA. Prior to freezing (−80 ◦ C), an aliquot of blood was transferred under a Class II Biosafety cabinet into a 2-mL Nalgene® cryovial (sterile, bulk-packed part no. 5012-0020, or similar) for storage until analysis. 3. Results and discussion 3.1. Preliminary experiments Different procedures have been proposed for the extraction of mercury species in biological samples for speciation purposes based on HPLC–ICP-MS [10,11] or GC–ICP-MS [18,19]. In general, protocols are based on acid [9,10] or basic extractions [18] mediums. However, most of these methodologies have several disadvantages when coupling with HPLC–ICP-MS. Moreover, as far as we know there are no papers dealing with mercury speciation in blood by using HPLC–ICP-MS. Firstly, as far as a compatible pH value for the reverse-phase column is concerned, a laborious procedure usually has to be adopted to adjust appropriate pH of the extracted solution prior to injection into the HPLC. Secondly, Hg species transformation might occur during sample preparation [20,21]. In order to avoid the aforementioned limitations, alternative extraction procedures have been suggested with reagents containing thiol ligands, such as mercaptoethanol [10], l-cysteine [10] or thiorea [24]. These procedures are associated with the use of microwave energy [25]. Alternatively, quantitative mercury extractions from hair samples have been demonstrated even in low acid conditions when associated with ultrasound energy [26]. The method of extraction evaluated here is based in part on a previous method described by Chiou et al. [10] for Hg speciation in fish samples. In that method, the authors have suggested the use of an extraction solution containing l-cysteine and 2mercaptoethanol in combination with microwave radiation. Thus, our preliminary experiments were carried out to explore the efficiency of using mercaptoethanol and l-cysteine for quantitative mercury extraction from hair samples with two basic differences from the method proposed by Chiou et al. [10] Firstly, we used ultrasound energy instead of microwave energy. Secondly, we also evaluated the use of a dilute solution of HCl (0.10%, v/v) in the extractant to accelerate the ultrasound extraction without a major change in the pH. The use of an ultrasound bath simplifies the method, since this system is much simpler and less expensive than commercial microwave systems.
3.3. Optimization of LC operating conditions After the optimization of mercury extraction from blood samples, we optimized the mobile phase composition. Different combinations of reagents in the mobile phase are usually recommended for the speciation of Hg in biological samples by HPLC–ICP-MS. Some authors recommend the use of l-cysteine and mercaptoethanol [10] while others recommend methanol, mercaptoethanol and ammonium acetate [11] or a mixture of l-cysteine, pyridine and methanol [27]. Our preliminary experiments demonstrated more promising results (time of separation, resolution, selectivity and sensitivity) for the mixture of mercaptoethanol, lcysteine, ammonium acetate and methanol. According to Chiou et al. [10] the retention time of mercury species increases with the increase in mercaptoethanol concentration in the mobile phase. We have observed the same results (data not shown). Thus, we fixed mercaptoethanol concentration at 0.05% (v/v) as a compromise between selectivity and time of analysis. Ammonium acetate was also fixed at 0.06 mol L−1 . This concentration is able to maintain a favorable pH at 6.7. On the other hand, different concentrations of methanol in the mobile phase were evaluated. For this study the concentrations of ammonium acetate, l-cysteine and mercaptoethanol were fixed at 0.06 mol L−1 , 0.05% (m/v) and 0.05% (v/v), respectively and the concentration of methanol was varied from 0.0% to 5% (v/v). A considerable increase in Hg sensitivity was observed with the increase in methanol concentration. Three mechanisms have been put forward to explain the above enhancement effect on signal intensities: (1) charge transfer reaction from C+ species to analyte atoms, (2) improvement in the nebulization transport of the sample, and (3) shift of the zone of maximum ion density [28]. Concentrations of methanol higher than 5% (v/v) were not evaluated, since they lead to plasma instability and an increase in carbon residues on cones. Thus the methanol concentration in the mobile phase was fixed at 5% (v/v) for further studies. Subsequent experiments were carried out to optimize the concentration of l-cysteine in the mobile phase. Separation of mercury species can take place based on the cysteine–mercury complexes on the polymeric-based C18 reverse-phase column. Concentrations of l-cysteine between 0.05% and 0.4% (m/v) were evaluated with the concentration of mercaptoethanol, ammonium acetate and methanol fixed at 0.05% (v/v), 0.06 mol L−1 and 5% (v/v), respectively. It has been observed that the higher the concentration of l-cysteine, the lower the retention time of the three mercury species and the higher the sensitivity for all mercury species. The separation of the three mercury species is achieved in less than 8 min with l-cysteine concentration fixed in 0.4% (m/v) compared to 35 min when 0.05% (m/v) l-cysteine is used in the mobile phase. Concentrations of l-cysteine higher than 0.4% (m/v) do not provide a complete separation between Met-Hg and Ino-Hg peaks. As a result, a solution containing 0.4% (m/v) l-cysteine, 0.05% (v/v) mercaptoethanol, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol was used as the mobile phase.
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Table 2 Speciation of mercury in the NIST 966 blood reference material. Found values are denoted as mean (SD), n = 3. Sample
Target values
LC–ICP-MS method
SRM
Ino-Hg concentration (g L−1 )
Total concentration (g L−1 )
Ino-Hg concentration (g L−1 )
Met-Hg concentration (g L−1 )
Total concentration (g L−1 )
NIST 966
14.87 ± 0.93
31.4 ± 1.7
14.8 (0.4)
16.5 (0.5)
31.3
Table 3 Mercury speciation in blood samples collected from riparians living in the Brazilian Amazon exposed to Met-Hg from fish consumption (values are denoted as mean (SD), n = 3). ND = not detected. Sample
Ino-Hg (g L−1 )
Met-Hg (g L−1 )
Et-Hg (g L−1 )
Total Hg Proposed method (g L−1 )
Total Hg [ICP-MS] [13] (g L−1 )
1 2 3 4 5 6 7 8 9 10
6.4 (1.1) 5.0 (0.2) 5.0 (0.3) 6.7 (1.1) 4.8 (0.6) 5.8 (0.8) 5.8(1.1) 4.2 (0.2) 2.0 (0.1) 2.3 (0.1)
70.7 (1.6) 47.3(0.1) 59.1 (0.2) 85.1 (0.7) 83.1 (0.2) 33.2 (1.4) 53.1 (1.7) 24.5 (0.6) 20.3 (0.4) 23.2 (1.4)
ND ND ND ND ND ND ND ND ND ND
77.1 52.4 64.1 91.8 88 39.0 59 28.7 22.3 25.5
76.8 (1.9) 52.9 (1.7) 66 (1.8) 103.3 (2) 86 (1) 39.8 (1.6) 58.8 (1.8) 28.2 (0.5) 22 (0.2) 24.1 (1.4)
3.4. Validation studies Validation of the proposed method was accomplished using NIST SRM 966 Bovine Blood. For additional validation, it was also analyzed 10 human blood specimens. Data obtained with the proposed method were compared to the results obtained using direct sample introduction for total mercury determination by ICP-MS. Results for NIST SRM 966 are shown in Table 2. Values found using the proposed method are in good agreement with established target values for the SRM 966. Moreover, no statistical differences were observed between the sum of the Hg species concentration
(proposed method) and the total Hg values found by an alternative method at 95% level on applying the t-test for the analysis of the 10 human blood samples (Table 3). Typical calibration graphics for methylmercury, ethylmercury and inorganic mercury are shown in Fig. 1 with a very good linearity. 3.5. Analytical parameters The LC–ICP-MS proposed method detection limit (3 SD) was 0.1 g L−1 and 0.25 g L−1 for methylmercury and inorganic mercury, respectively. Typical within-day precision was always lower
Fig. 1. Calibration curves for different mercury species by applying the proposed LC–ICP-MS method. Standards of each mercury form were prepared from 0.0 g L−1 to 20 g L−1 . Mobile phase consisting of 0.4% (m/v) l-cysteine, 0.05% (v/v) mercaptoethanol, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol. For other conditions see Table 1.
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mercury was extracted according to the proposed procedure and left at room temperature until analysis. Then, samples were analyzed after the first hour (T0), 3 h later (T1), 8 h later (T2), 24 h later (T3) and 4 days later (T4). Our results demonstrated that mercury species present in this certified material (methylmercury and inorganic mercury) can be extracted and left at room temperature up to 4 days before analysis. Since this blood reference material does not contain ethylmercury, additional experiment was carried out with an ordinary blood (human blood), divided in four fractions of 250 L and each fraction spiked with ethylmercury to contain 10 g L−1 . The proposed method of extraction was then applied and the resulted extracted solutions were left at room temperature at the same time of the previous experiment with the NIST 966. Contrary to methylmercury and inorganic mercury recovery of ethylmercury was lower than 30% even when the speciation analysis was performed just after the blood was spiked. Moreover, this loss was proportional to an increase on the amount of inorganic mercury. Since ethylmercury is stable in the standards (at room temperature and after 15 min of ultrasound energy application), it means that ethylmercury is converted to inorganic mercury in blood for some blood component with a mechanism not yet known. 3.9. In vivo conversion of ethylmercury to inorganic mercury
Fig. 2. Chromatograms showing the separation of mercury species in: (a) standards containing 10 g L−1 of each mercury compound and (b) human blood (volunteer from fish-eating communities of the Amazon region). Mobile phase consisting of 0.4% (m/v) cysteine, 0.05% (v/v) mercaptoethanol, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol. For other conditions see Table 1.
Based on the preliminary results with ethylmercury in blood, we did a further experiment with exposed rats. Two male Wistar rats weighting 200 g each were treated by gavage with ethylmercury solution during 3 days (100 g kg−1 day−1 ) and the blood was collected from the animals before the treatment (blank) and in the fourth day after the beginning of the treatment. Then, the determination of three mercury species (inorganic, methylmer-
than 7.0% (NIST SRM 966), while between-day precision was <12.0% RSD (NIST SRM 966) for both methylmercury and inorganic mercury determinations. 3.6. Method application for the speciation of mercury in blood samples collected from fish-eating communities Human blood specimens were collected from 10 volunteers living in the Brazilian Amazon and exposed to high levels of methylmercury from fish consumption. Results are shown in Table 3. Only methylmercury and inorganic mercury were identified in these samples (Fig. 2). Moreover, total mercury levels found with the proposed method as a sum of inorganic and methylmercury are in good agreement with Hg values found by ICP-MS. 3.7. Stability of blood samples after collection for mercury speciation After the speciation analysis of blood samples collected from fish-eating communities, they were left at −80 ◦ C during 6 months to check the long-term stability of samples after collection. After this period, samples were re-analyzed by the proposed procedure. There were no statistical differences between the values in the two periods of analysis, demonstrating that samples are stable after collection at least 6 months at −80 ◦ C. 3.8. Stability of mercury species after extraction To verify the stability of the mercury species after extraction, a time study of sample storage at room temperature was carried out. For this experiment the SRM NIST 966 reference material was divided into four different fractions of 250 L. From each fraction,
Fig. 3. Chromatograms showing the separation of mercury species in blood of: (a) rat before ethylmercury exposure (control) and (b) exposed to 100 g kg−1 day−1 of ethylmercury during 3 days. For experimental conditions see Table 1.
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cury and ethylmercury) was carried out by applying the proposed method. Fig. 3 shows the chromatogram of one of the rat’s blood before (Fig. 3a) and after ethylmercury exposure (Fig. 3b). As can be observed two peaks were obtained in the blood of the exposed animal, one predominant of inorganic mercury and a very small peak of methylmercury. It demonstrates the in vivo conversion of ethylmercury largely to inorganic mercury and to much less extent to methylmercury. This finding is extremely relevant in the evaluation of thimerosal exposed populations, since, thimerosal, a preservative in many currently marketed vaccines throughout the world, releases ethyl mercury radical as the active species. 4. Conclusion A simple method has been developed for mercury speciation in blood samples based on LC–ICP-MS. Sample preparation procedure is very fast and simple with a quantitative extraction of mercury in 15 min. In addition, the number of handling steps, sample preparation and analysis time, as well as potential sources of analytical errors, is reduced. The method was successfully applied for the speciation of mercury in blood samples collected from volunteers exposed to methylmercury by fish consumption. Finally, it was demonstrated the ethylmercury is practically fully converted in vivo to inorganic mercury and in much lees extend to methylmercury and then the inorganic form. It brings new insights to the toxicological aspects and mechanisms of thimerosal containing vaccines. Acknowledgments The authors are grateful to Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support and fellowships.
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Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure Jairo L. Rodrigues, Samuel S. de Souza, Vanessa C. de Oliveira Souza, Fernando Barbosa Jr. ∗ Laboratório de Toxicologia e Essencialidade de Metais, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
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Article history: Received 16 July 2009 Received in revised form 31 August 2009 Accepted 1 September 2009 Available online 9 September 2009 Keywords: ICP-MS Speciation Blood samples Mercury Sample preparation Methylmercury Thimerosal Liquid chromatography
a b s t r a c t Despite the necessity to differentiate chemical species of mercury in clinical specimens, there are a limited number of methods for this purpose. Then, this paper describes a simple method for the determination of methylmercury and inorganic mercury in blood by using liquid chromatography with inductively coupled mass spectrometry (LC–ICP-MS) and a fast sample preparation procedure. Prior to analysis, blood (250 L) is accurately weighed into 15-mL conical tubes. Then, an extractant solution containing mercaptoethanol, l-cysteine and HCl was added to the samples following sonication for 15 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 5 min on a C18 reverse-phase column with a mobile phase containing 0.05% (v/v) mercaptoethanol, 0.4% (m/v) l-cysteine, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol. The method detection limits were found to be 0.25 g L−1 and 0.1 g L−1 for inorganic mercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). The proposed method was also applied to the speciation of mercury in blood samples collected from fish-eating communities and from rats exposed to thimerosal. With the proposed method there is a considerable reduction of the time of sample preparation prior to speciation of Hg by LC–ICP-MS. Finally, after the application of the proposed method, we demonstrated an interesting in vivo ethylmercury conversion to inorganic mercury. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Speciation analysis of clinical samples is gradually becoming more widely accepted for nutritional and/or toxicological purposes. According to International Union of Pure and Applied Chemistry (IUPAC), speciation analysis is defined as the analytical process of identifying and/or measuring quantities of one or more individual chemical forms in a sample, and speciation of an element is defined as the distribution of an element among defined chemical species in a system [1,2]. Mercury (Hg) is one of the most hazardous pollutants in the environment. It exists in three basic forms: elemental mercury (Hg0 ) known as metallic mercury, inorganic mercury compounds (Ino-Hg), primarily mercuric chloride, and organic mercury, primarily methylmercury (Met-Hg) [3,4]. Organic forms are more toxic than inorganic [4]. The main sources of human exposure to organic mercurials, mainly in the form of methylmercury (Met-Hg) or ethylmercury
∗ Corresponding author. Tel.: +55 16 36024701. E-mail address: [email protected] (F. Barbosa Jr.). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.09.001
(Et-Hg), are the consumption of fish [4] and vaccines containing thimerosal, respectively [5]. On the other hand, the most common forms of exposure to inorganic forms are by inhalation of Hg vapor released from dental amalgams or from gold mining activities [4]. Measurements of Hg in both blood and hair are used to determine whether adverse health effects are likely to occur. The contents of mercury species in blood may represent a cumulative exposure from the daily diet and/or occupational environment exposure. However, despite the difference in toxicity related to the form of mercury, total mercury concentration (THg) in blood is usually used to estimate the clinical outcomes after mercury exposure. Moreover, the proportion between chemical forms of mercury in blood may vary among individuals. This makes it essential to have analytical methods, based on speciation analysis, which can differentiate between chemical forms in blood to diagnose risks of toxicity. The most effective instrumental-based techniques for chemical speciation analysis rely on the use of chromatography (mainly gas chromatography (GC) [6–9] or liquid chromatography (LC)) [10–12] coupled to a specific and sensitive detector, such as inductively coupled plasma mass spectrometry (ICP-MS). Compared with GC, LC is
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the preferred separation technique used for mercury speciation, because the mercury species do not need to be derived to volatile compounds before HPLC separation. Although several methods have been developed for measuring mercury in blood samples [13–17], there is merely a few proposing speciation analysis [18,19]. Moreover, all of them suggested very tedious and time-consuming sample preparation procedures. Moreover, artifact formation during the sample preparation step is a potential source of error [20,21]. On the other hand, clinical laboratories must cope with an increasing demand for trace element analysis in body fluids and tissues in response to increasing concern for occupational and environmental exposure to mercury. Thus, fast sample preparation procedures with minimal handling are extremely desirable in routine analysis. The aim of this paper was therefore to evaluate a simple method for methylmercury and inorganic mercury determination in blood by high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) with a fast sample preparation procedure prior to analysis. The method was then applied for speciation of mercury in blood samples collected from a Brazilian riparians, living in the Amazon region, exposed to mercury from fish consumption. It was also demonstrated for the first time that ethylmercury is in vivo converted to inorganic mercury, making the determination of the inorganic form of mercury in blood relevant for thimerosal exposed populations.
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Table 1 Liquid chromatography and ICP-MS operating conditions for Hg speciation in blood samples. LC conditions Column Pre-column (Guard column)
Mobile phase flow rate Sample loop Measurement
C18 (5 m, 150 mm × 4 mm) RP18 (7 m, 15 mm × 3.2 mm) 0.05% (v/v) mercaptoethanol 0.4% (m/v) l-cysteine 0.06 mol L−1 ammonium acetate 5% (v/v) methanol 1 mL min−1 100 L Peak high
ICP-MS experimental conditions Radio frequency power (W) Scan mode Nebulizer gas flow (L min−1 ) Resolution (amu) Replicates Isotopes
1200 Peak hopping 0.58 0.7 3 202 Hg
Mobile phase
2.4. Reagents
A PerkinElmer model L-200 LC pump, six-port injector (Rheodyne 9725) with a reverse-phase column (C18, 5 m, 150 mm × 4 mm, Brownlee Columns PerkinElmer, USA) and a precolumn (guard column) RP18 (7 m, 15 mm × 3.2 mm, Brownlee Columns PerkinElmer, USA) comprised the LC system. Samples were loaded with a syringe into a 100-L sample loop. All separations were performed at room temperature under isocratic conditions. The isocratic mobile phase was 0.05% (v/v) mercaptoethanol, 0.4% (m/v) l-cysteine, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol. The flow rate was 1.0 mL min−1 . The effluent from the LC column was directly connected to the nebulizer with PEEK tubing (1.59 mm o.d.) and a low dead volume PEEK connector. Data evaluation was performed using Chromera® software supplied with the instrument, and quantification was based on peak high by external calibration. The optimum experimental conditions for both ICP-MS and LC are given in Table 1.
All reagents used were of analytical grade and the solutions were prepared using high-purity water with a resistivity of 18.2 M cm, obtained from a Milli-Q Plus water purification system (Millipore, Bedford, MA, USA). Nitric acid (Merck, Darmstadt, Germany), was doubly distilled in a quartz sub-boiling apparatus (Kürner Analysentechnik, Rosenheim, Germany). A clean laboratory and laminar-flow hood capable of producing class 100 were used for preparing solutions and samples. All solutions were stored in high-density polyethylene bottles. Plastic bottles and glassware materials were cleaned by soaking in 10% (v/v) HNO3 for 24 h, rinsed five times with Milli-Q water and dried in a class 100 laminar-flow hood before use. All operations were performed on a clean bench. A 10-mg L−1 standard solution of inorganic mercury was obtained from PerkinElmer (PerkinElmer, Norwalk, CT). A 1000mg L−1 standard solution of methylmercury chloride (CH3 HgCl) and 1000-mg L−1 standard solution of ethylmercury chloride (CH3 CH2 HgCl) in water were obtained from Alfa Aesar. Analytical calibration standards of mercury species were prepared daily over the range of 0.0–20.0 gL−1 for the LC–ICP-MS method by suitable serial dilutions of the stock solution in the mobile phase. Additional chemicals for the speciation studies were HPLC grade methanol (99.9%, v/v) and mercaptoethanol (Sigma–Aldrich, USA), l-cysteine (Fluka, Japan). Ammonium acetate (99.99%) was obtained from Aldrich Chemical Company (Milwaukee, USA). For the ICP-MS method for total mercury determination, Rh and Au were each diluted from 1000 mg L−1 stock standards (Assurance grade; Spex Certiprep® ). Triton® X-100 was obtained from the Sigma–Aldrich Co. (Sigma Ultra Grade, Sigma–Aldrich Co., St. Louis, MO, USA) and was diluted to 10% (v/v) with double-deionized water. A rinse solution consisting of 0.005% (v/v) Triton X-100® and 1 mg L−1 Au in 2% (v/v) nitric acid was prepared before each run.
2.3. Measurements of total mercury in blood
2.5. Sample preparation for speciation analysis
The method used for the determination of total mercury in blood samples was based on a previous publication described by Palmer et al. [13]. Briefly, blood specimens were diluted 1 + 49 into a 15mL polypropylene Falcon® tube (Blue MaxTM Jr., Becton Dickinson) with a diluent solution containing 0.5% (v/v) double-distilled HNO3 , 25 g L−1 Rh as internal standard, 1 mg L−1 Au to control Hg memory effects in the spray-chamber, and 0.005% (v/v) Triton® X-100 and analyzed by ICP-MS.
Blood samples (250 L) were placed in 15 mL polypropylene test tubes with 4.75 mL of a solution containing 0.10% (v/v) HCl + 0.05% (m/v) l-cysteine + 0.10% (v/v) 2-mercaptoethanol and then sonicated for 15 min in an ultrasonic bath 1400 A (UNIQUE, Brazil). The resulting solution was centrifuged and then filtered through 0.20 m Nylon® filters (Millipore, USA). Sample extraction was performed in triplicate and extraction blanks were prepared in the same manner.
2. Material and methods 2.1. Instruments and apparatus All measurements were made with an ICP-MS (Elan DRC II PerkinElmer, Norwalk, CT) for total mercury determination and for speciation. 2.2. Measuring of mercury species
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2.6. Standard reference materials and ordinary blood specimens
3.2. Evaluation of the time of ultrasound extraction
Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, MD). For additional validation purposes, human blood specimens (n = 10) collected as part of another research study involving Hg exposure in the Brazilian Amazon region were available for method comparison purposes with identifiers removed [22,23]. These specimens had been obtained with informed consent from human subjects in accordance with procedures approved by our Institutional Review Board.
For this experiment, it has been evaluated the extractor solution containing 0.10% (v/v) HCl + 0.05% (m/v) l-cysteine + 0.10% (v/v) 2mercaptoethanol and different times of ultrasound energy (from 0 to 30 min). Different aliquots (250 L) of the SRM NIST 966 were used for the experiment. Quantitative extraction of Hg (>95%) was observed for the NIST 966 blood by using the proposed extraction solution and 15 min of ultrasound energy. Then, for the subsequent experiments, mercury species were extracted from the blood samples by using the solution containing 0.10% (v/v) HCl + 0.05% (m/v) l-cysteine + 0.10% (v/v) 2-mercaptoethanol and 15 min of ultrasound energy.
2.7. Blood specimen collection Venous blood was collected into pre-screened, 3-mL draw lavender-capped BD Vacutainer® blood collection tubes containing K2EDTA anticoagulant (Becton Dickinson, Franklin Lakes, NJ, USA; part no. 367856). These tubes were used with BD Vacutainer® EclipseTM 22 gauge, 1–1.25 in. blood collection needles (Becton Dickinson; part no. 368608). After blood was drawn into the Vacutainer® , the tube was inverted several times to mix blood with K2EDTA. Prior to freezing (−80 ◦ C), an aliquot of blood was transferred under a Class II Biosafety cabinet into a 2-mL Nalgene® cryovial (sterile, bulk-packed part no. 5012-0020, or similar) for storage until analysis. 3. Results and discussion 3.1. Preliminary experiments Different procedures have been proposed for the extraction of mercury species in biological samples for speciation purposes based on HPLC–ICP-MS [10,11] or GC–ICP-MS [18,19]. In general, protocols are based on acid [9,10] or basic extractions [18] mediums. However, most of these methodologies have several disadvantages when coupling with HPLC–ICP-MS. Moreover, as far as we know there are no papers dealing with mercury speciation in blood by using HPLC–ICP-MS. Firstly, as far as a compatible pH value for the reverse-phase column is concerned, a laborious procedure usually has to be adopted to adjust appropriate pH of the extracted solution prior to injection into the HPLC. Secondly, Hg species transformation might occur during sample preparation [20,21]. In order to avoid the aforementioned limitations, alternative extraction procedures have been suggested with reagents containing thiol ligands, such as mercaptoethanol [10], l-cysteine [10] or thiorea [24]. These procedures are associated with the use of microwave energy [25]. Alternatively, quantitative mercury extractions from hair samples have been demonstrated even in low acid conditions when associated with ultrasound energy [26]. The method of extraction evaluated here is based in part on a previous method described by Chiou et al. [10] for Hg speciation in fish samples. In that method, the authors have suggested the use of an extraction solution containing l-cysteine and 2mercaptoethanol in combination with microwave radiation. Thus, our preliminary experiments were carried out to explore the efficiency of using mercaptoethanol and l-cysteine for quantitative mercury extraction from hair samples with two basic differences from the method proposed by Chiou et al. [10] Firstly, we used ultrasound energy instead of microwave energy. Secondly, we also evaluated the use of a dilute solution of HCl (0.10%, v/v) in the extractant to accelerate the ultrasound extraction without a major change in the pH. The use of an ultrasound bath simplifies the method, since this system is much simpler and less expensive than commercial microwave systems.
3.3. Optimization of LC operating conditions After the optimization of mercury extraction from blood samples, we optimized the mobile phase composition. Different combinations of reagents in the mobile phase are usually recommended for the speciation of Hg in biological samples by HPLC–ICP-MS. Some authors recommend the use of l-cysteine and mercaptoethanol [10] while others recommend methanol, mercaptoethanol and ammonium acetate [11] or a mixture of l-cysteine, pyridine and methanol [27]. Our preliminary experiments demonstrated more promising results (time of separation, resolution, selectivity and sensitivity) for the mixture of mercaptoethanol, lcysteine, ammonium acetate and methanol. According to Chiou et al. [10] the retention time of mercury species increases with the increase in mercaptoethanol concentration in the mobile phase. We have observed the same results (data not shown). Thus, we fixed mercaptoethanol concentration at 0.05% (v/v) as a compromise between selectivity and time of analysis. Ammonium acetate was also fixed at 0.06 mol L−1 . This concentration is able to maintain a favorable pH at 6.7. On the other hand, different concentrations of methanol in the mobile phase were evaluated. For this study the concentrations of ammonium acetate, l-cysteine and mercaptoethanol were fixed at 0.06 mol L−1 , 0.05% (m/v) and 0.05% (v/v), respectively and the concentration of methanol was varied from 0.0% to 5% (v/v). A considerable increase in Hg sensitivity was observed with the increase in methanol concentration. Three mechanisms have been put forward to explain the above enhancement effect on signal intensities: (1) charge transfer reaction from C+ species to analyte atoms, (2) improvement in the nebulization transport of the sample, and (3) shift of the zone of maximum ion density [28]. Concentrations of methanol higher than 5% (v/v) were not evaluated, since they lead to plasma instability and an increase in carbon residues on cones. Thus the methanol concentration in the mobile phase was fixed at 5% (v/v) for further studies. Subsequent experiments were carried out to optimize the concentration of l-cysteine in the mobile phase. Separation of mercury species can take place based on the cysteine–mercury complexes on the polymeric-based C18 reverse-phase column. Concentrations of l-cysteine between 0.05% and 0.4% (m/v) were evaluated with the concentration of mercaptoethanol, ammonium acetate and methanol fixed at 0.05% (v/v), 0.06 mol L−1 and 5% (v/v), respectively. It has been observed that the higher the concentration of l-cysteine, the lower the retention time of the three mercury species and the higher the sensitivity for all mercury species. The separation of the three mercury species is achieved in less than 8 min with l-cysteine concentration fixed in 0.4% (m/v) compared to 35 min when 0.05% (m/v) l-cysteine is used in the mobile phase. Concentrations of l-cysteine higher than 0.4% (m/v) do not provide a complete separation between Met-Hg and Ino-Hg peaks. As a result, a solution containing 0.4% (m/v) l-cysteine, 0.05% (v/v) mercaptoethanol, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol was used as the mobile phase.
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Table 2 Speciation of mercury in the NIST 966 blood reference material. Found values are denoted as mean (SD), n = 3. Sample
Target values
LC–ICP-MS method
SRM
Ino-Hg concentration (g L−1 )
Total concentration (g L−1 )
Ino-Hg concentration (g L−1 )
Met-Hg concentration (g L−1 )
Total concentration (g L−1 )
NIST 966
14.87 ± 0.93
31.4 ± 1.7
14.8 (0.4)
16.5 (0.5)
31.3
Table 3 Mercury speciation in blood samples collected from riparians living in the Brazilian Amazon exposed to Met-Hg from fish consumption (values are denoted as mean (SD), n = 3). ND = not detected. Sample
Ino-Hg (g L−1 )
Met-Hg (g L−1 )
Et-Hg (g L−1 )
Total Hg Proposed method (g L−1 )
Total Hg [ICP-MS] [13] (g L−1 )
1 2 3 4 5 6 7 8 9 10
6.4 (1.1) 5.0 (0.2) 5.0 (0.3) 6.7 (1.1) 4.8 (0.6) 5.8 (0.8) 5.8(1.1) 4.2 (0.2) 2.0 (0.1) 2.3 (0.1)
70.7 (1.6) 47.3(0.1) 59.1 (0.2) 85.1 (0.7) 83.1 (0.2) 33.2 (1.4) 53.1 (1.7) 24.5 (0.6) 20.3 (0.4) 23.2 (1.4)
ND ND ND ND ND ND ND ND ND ND
77.1 52.4 64.1 91.8 88 39.0 59 28.7 22.3 25.5
76.8 (1.9) 52.9 (1.7) 66 (1.8) 103.3 (2) 86 (1) 39.8 (1.6) 58.8 (1.8) 28.2 (0.5) 22 (0.2) 24.1 (1.4)
3.4. Validation studies Validation of the proposed method was accomplished using NIST SRM 966 Bovine Blood. For additional validation, it was also analyzed 10 human blood specimens. Data obtained with the proposed method were compared to the results obtained using direct sample introduction for total mercury determination by ICP-MS. Results for NIST SRM 966 are shown in Table 2. Values found using the proposed method are in good agreement with established target values for the SRM 966. Moreover, no statistical differences were observed between the sum of the Hg species concentration
(proposed method) and the total Hg values found by an alternative method at 95% level on applying the t-test for the analysis of the 10 human blood samples (Table 3). Typical calibration graphics for methylmercury, ethylmercury and inorganic mercury are shown in Fig. 1 with a very good linearity. 3.5. Analytical parameters The LC–ICP-MS proposed method detection limit (3 SD) was 0.1 g L−1 and 0.25 g L−1 for methylmercury and inorganic mercury, respectively. Typical within-day precision was always lower
Fig. 1. Calibration curves for different mercury species by applying the proposed LC–ICP-MS method. Standards of each mercury form were prepared from 0.0 g L−1 to 20 g L−1 . Mobile phase consisting of 0.4% (m/v) l-cysteine, 0.05% (v/v) mercaptoethanol, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol. For other conditions see Table 1.
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mercury was extracted according to the proposed procedure and left at room temperature until analysis. Then, samples were analyzed after the first hour (T0), 3 h later (T1), 8 h later (T2), 24 h later (T3) and 4 days later (T4). Our results demonstrated that mercury species present in this certified material (methylmercury and inorganic mercury) can be extracted and left at room temperature up to 4 days before analysis. Since this blood reference material does not contain ethylmercury, additional experiment was carried out with an ordinary blood (human blood), divided in four fractions of 250 L and each fraction spiked with ethylmercury to contain 10 g L−1 . The proposed method of extraction was then applied and the resulted extracted solutions were left at room temperature at the same time of the previous experiment with the NIST 966. Contrary to methylmercury and inorganic mercury recovery of ethylmercury was lower than 30% even when the speciation analysis was performed just after the blood was spiked. Moreover, this loss was proportional to an increase on the amount of inorganic mercury. Since ethylmercury is stable in the standards (at room temperature and after 15 min of ultrasound energy application), it means that ethylmercury is converted to inorganic mercury in blood for some blood component with a mechanism not yet known. 3.9. In vivo conversion of ethylmercury to inorganic mercury
Fig. 2. Chromatograms showing the separation of mercury species in: (a) standards containing 10 g L−1 of each mercury compound and (b) human blood (volunteer from fish-eating communities of the Amazon region). Mobile phase consisting of 0.4% (m/v) cysteine, 0.05% (v/v) mercaptoethanol, 0.06 mol L−1 ammonium acetate and 5% (v/v) methanol. For other conditions see Table 1.
Based on the preliminary results with ethylmercury in blood, we did a further experiment with exposed rats. Two male Wistar rats weighting 200 g each were treated by gavage with ethylmercury solution during 3 days (100 g kg−1 day−1 ) and the blood was collected from the animals before the treatment (blank) and in the fourth day after the beginning of the treatment. Then, the determination of three mercury species (inorganic, methylmer-
than 7.0% (NIST SRM 966), while between-day precision was <12.0% RSD (NIST SRM 966) for both methylmercury and inorganic mercury determinations. 3.6. Method application for the speciation of mercury in blood samples collected from fish-eating communities Human blood specimens were collected from 10 volunteers living in the Brazilian Amazon and exposed to high levels of methylmercury from fish consumption. Results are shown in Table 3. Only methylmercury and inorganic mercury were identified in these samples (Fig. 2). Moreover, total mercury levels found with the proposed method as a sum of inorganic and methylmercury are in good agreement with Hg values found by ICP-MS. 3.7. Stability of blood samples after collection for mercury speciation After the speciation analysis of blood samples collected from fish-eating communities, they were left at −80 ◦ C during 6 months to check the long-term stability of samples after collection. After this period, samples were re-analyzed by the proposed procedure. There were no statistical differences between the values in the two periods of analysis, demonstrating that samples are stable after collection at least 6 months at −80 ◦ C. 3.8. Stability of mercury species after extraction To verify the stability of the mercury species after extraction, a time study of sample storage at room temperature was carried out. For this experiment the SRM NIST 966 reference material was divided into four different fractions of 250 L. From each fraction,
Fig. 3. Chromatograms showing the separation of mercury species in blood of: (a) rat before ethylmercury exposure (control) and (b) exposed to 100 g kg−1 day−1 of ethylmercury during 3 days. For experimental conditions see Table 1.
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cury and ethylmercury) was carried out by applying the proposed method. Fig. 3 shows the chromatogram of one of the rat’s blood before (Fig. 3a) and after ethylmercury exposure (Fig. 3b). As can be observed two peaks were obtained in the blood of the exposed animal, one predominant of inorganic mercury and a very small peak of methylmercury. It demonstrates the in vivo conversion of ethylmercury largely to inorganic mercury and to much less extent to methylmercury. This finding is extremely relevant in the evaluation of thimerosal exposed populations, since, thimerosal, a preservative in many currently marketed vaccines throughout the world, releases ethyl mercury radical as the active species. 4. Conclusion A simple method has been developed for mercury speciation in blood samples based on LC–ICP-MS. Sample preparation procedure is very fast and simple with a quantitative extraction of mercury in 15 min. In addition, the number of handling steps, sample preparation and analysis time, as well as potential sources of analytical errors, is reduced. The method was successfully applied for the speciation of mercury in blood samples collected from volunteers exposed to methylmercury by fish consumption. Finally, it was demonstrated the ethylmercury is practically fully converted in vivo to inorganic mercury and in much lees extend to methylmercury and then the inorganic form. It brings new insights to the toxicological aspects and mechanisms of thimerosal containing vaccines. Acknowledgments The authors are grateful to Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support and fellowships.
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