Accred Qual Assur (2009) 14:263–267 DOI 10.1007/s00769-009-0509-8
GENERAL PAPER
Development of a reference measurement procedure for the determination of methylmercury in fish products Se´bastien Sannac Æ Paola Fisicaro Æ Guillaume Labarraque Æ Florence Pannier Æ Martine Potin-Gautier
Received: 3 November 2008 / Accepted: 3 March 2009 / Published online: 21 March 2009 Ó Springer-Verlag 2009
Abstract The development of an analytical procedure for speciation analysis of methylmercury in fish products is presented. The method is based on high-performance liquid chromatography hyphenated to inductively coupled plasmamass spectrometry. The metrological approach is stressed out in this paper, in order to provide reliable and comparable results. A complete uncertainty budget has been evaluated and the method has been validated by the use of a certified reference material. Moreover, the detection could rely on the isotope dilution mass spectrometry, a powerful strategy capable of highly accurate results traceable to the ‘‘Syste`me International d’Unite´s’’ and recognised by the ‘‘Comite´ Consultatif pour la Quantite´ de Matie`re’’ as a primary method of measurement. Keywords Speciation analysis Methylmercury Species-specific isotope dilution
Introduction Speciation analysis, i.e. the identification, quantification and characterisation of the chemical forms of a given
Presented at MEFNM 2008, September 2008, Budapest, Hungary. S. Sannac P. Fisicaro (&) G. Labarraque Laboratoire national de me´trologie et d’essais (LNE), 1 rue Gaston Boissier, 75015 Paris, France e-mail:
[email protected] S. Sannac F. Pannier M. Potin-Gautier Laboratoire de Chimie Analytique, Bio-Inorganique et Environnement (UMR 5254 IPREM), Universite´ de Pau et des Pays de l’Adour/CNRS, He´lioparc, 2 av. Pierre Angot, 64053 Pau, France
element [1], is one of the key challenges of modern analytical chemistry. Mechanisms of accumulation, storage, or expulsion within an organism are, for instance, strongly dependent on the chemical species [2]. Undoubtedly, one of the most important applications of speciation is to be found in the area of toxicology. In fact, information on the total mass fraction of an element is very often not sufficient to provide information related to its toxicity [2]. Among the heavy metals, mercury is one of the most studied environmental pollutants. This is largely the result of its high toxicity and mobility in the environment. Due to the ability to travel over long distances in the atmosphere as gaseous elemental species, mercury is regarded as a ‘‘global pollutant’’ [3]. The high toxicity of mercury is given, inter alia, by its methylated form, methylmercury (hereafter MeHg?), which is widely recognised as a neurotoxin affecting humans [3]. The main pathway of contamination of the humans with mercury is through the nutrition. In particular, the great ability to be bioaccumulated in the aquatic food chain leads to considerably elevated levels of MeHg? in aquatic organisms in higher levels of the trophic chain, despite nearly immeasurable quantities of mercury in the water (bioconcentration factors are usually between 10 000 and 100 000) [3]. From the edible portions of fish, total Hg mass fractions higher than 1200 lg kg-1 can be found [3]. Consequently, as the greater proportion of Hg in fish is present as methylmercury, the consumption of 200 g of such fish can easily exceed the recommended limitation of MeHg? fixed at 1.6 lg per kg body mass per week [4]. Regulatory authorities are expected to measure concentrations of contaminants in foodstuffs, as it is the case in EU member states [5]. But the simple determination of
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total amount cannot be sufficient for fully judging its impact on the human health. In particular, the methylation of metals generally increases their toxicity by rendering them more lipid soluble and facilitating their crossing of lipid barriers such as the cell membrane or blood–tissue (e.g. blood–brain) barriers [2]. Therefore, reliable results in the assessment of methylmercury will be needed in the future. For the separation of mercury species, several methods have being proposed up to now based on the use of gas chromatography (GC) [6]. However, strong and thermally stable derivatives are needed, thus involving tedious and time consuming derivatisation and cleanup procedures. On the other hand, high-performance liquid chromatography (HPLC) offers an interesting alternative to GC providing a simplified and less time consuming sample preparation [7–9]. The aim of this study was the establishment of the metrological traceability to the International System of units (SI) of the entire measurement procedure, in order to provide reliable and comparable results. The implemented assay procedure could rely on the isotope dilution mass spectrometry (IDMS), a powerful strategy capable of highly accurate results traceable to the SI and recognised by the ‘‘Comite´ Consultatif pour la Quantite´ de Matie`re’’ (CCQM) as a primary method of measurement. The analysis of mercury species was achieved by HPLC hyphenated to inductively coupled plasma-mass spectrometry (ICP-MS). The optimisation of an extraction procedure in order to decrease the extraction duration and to limit contamination through reactants was also investigated. Finally, the procedure for the determination of MeHg? in fish matrices was validated by the use of a tuna certified reference material (CRM) and the establishment of a complete uncertainty budget. Hence, this procedure meets the criteria of a reference measurement procedure as specified by the International Vocabulary of Metrology (VIM) [10] and can be seen as an alternative to the mostly used method based on GC-ICP-MS [11, 12].
Experimental Reagents and samples Ultra-pure water was obtained from a Milli-Q system (Millipore Co., USA). Hydrochloric acid was in the range of Suprapur chemicals (Merck, Germany). Mercury species standards and reagents for extraction solvents and the HPLC mobile phase were purchased from Sigma-Aldrich (France). Isotopically labelled compound of MeHg?, enriched in isotope 200, was purchased from Applied Isotope
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Technologies (USA). IRMM-639, mercury certified for its isotopic composition, was obtained from Institute for Reference Materials and Measurements (IRMM, Belgium). The tests have been performed on a certified reference material (CRM) of freeze-dried tuna (BCR-463) certified for the total amount of mercury and the amount of MeHg? (IRMM). Instrumentation Sample preparation Water bath for incubation with magnetic stirring and ultrasonic bath were purchased from VWR (France). Microwave assisted extraction was performed with an Ethos 900 (Milestone, Italy). HPLC The chromatographic system was the SpectraSYSTEM provided by Thermo Fisher Scientific (USA). Separation was performed on a reverse phase C18 column (4 lm, 4.69150 mm, Synergi Hydro-RP, Phenomenex) after formation of a complex between the mercury in its different species and a sulphur compound, cysteine. The mobile phase was adapted from the literature [9] and was composed of two reagents at the following mass concentrations: 500 mg dm-3 L-Cysteine (L-Cys) and 500 mg dm-3 L-Cys, HCl, H2O, then its pH value was fixed at 2.3 by drop-wise addition of HCl (4 mol dm-3). 50 ll samples were injected and the flow was set at 1 ml min-1. ICP-MS The ICP-MS instrument was a PQ-Excell (Thermo Fisher Scientific), equipped with a quadrupole analyser to sort the mass. Table 1 recapitulates general settings for the ICP-MS. Extraction procedures Different extraction procedures of mercury species were tested on a tuna fish CRM. The protocol is based on a thiolcontaining reagent (cysteine) known to bind tightly mercury [13], as developed by Hight et al. [9]. Table 1 General settings of ICP-MS RF Power Plasma gas
1350 W 14 L min-1
Auxiliary
0.80 L min-1
Nebulization
0.98–1.03 L min-1
m/z Monitored
199, 200, 201 and 202
Dwell time
250 ms
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Extraction recoveries were evaluated by using three different approaches: (1) incubation of samples (in amber glass vials) for 2 h at 80 °C in a water bath and using magnetic stirring; (2) using an ultrasonic bath with different exposition times (15 min to 1 h) with manual stirring every 15 min and (3) microwave assisted extraction (at different powers and exposition times) in a closed system with Teflon vessel. In all procedures 40 mL of reagent were used to leach out mercury from 200 mg of sample except for the microwave assisted extraction where 150 mg of fish were extracted with 20 mL of reagent. Isotope dilution The selected extraction procedure was applied with the use of isotope dilution for the determination of methylmercury mass fraction. The double ID was involved as described previously [14]. The addition of the methylmercury spike (enriched in isotope 200Hg) was calculated to obtain a ratio between 202 Hg/200Hg equal to 0.8.
Results and discussion Optimisation of the extraction solvent Hight et al. [9] developed an extraction method with a media composed of 10 g dm-3 L-Cys during 2 h at 80 °C. This protocol was efficient but the use of such high amount of cysteine may contaminate samples with inorganic mercury (Hg2?) present in the reagent as shown in Fig. 1 where Hg2? could be detected for 50 lL injected. Therefore, the method was first adapted by using as the extraction media the chromatographic mobile phase (which
contains ten times lower amount of L-Cys) and incubation in a water bath at 80 °C for 2 h. As shown from the chromatogram presented in Fig. 2, only two species of Hg could be detected: Hg2? and MeHg?. Their quantification was first performed by external calibration (Table 2). The obtained mass fraction of methylmercury is in the range defined by the certified values, allowing us to assume that this protocol is efficient for quantitative extraction of MeHg?. Moreover, as the sum of species is compatible with the certified mass fraction for total mercury, the extraction of mercury can be also considered as quantitative for total mercury. In the further experiments, only the mobile phase was thus used as extraction solvent. Optimisation of the extraction time The improvement of the duration of the sample treatment was investigated. In order to check the extraction efficiency, the amount of MeHg? was chosen instead of the total amount of Hg due to the risk of species evolution during the extraction protocol. An extraction percentage of MeHg? was obtained by calculating the ratio between the determined and the certified values. For each test, three replicates were performed. The first series of tests allowed evaluating the efficiency of ultrasonic bath for different extraction times and the microwave field for 6 min at 70 W with respect to former results obtained with the water bath. Results are reported in Fig. 3 where the error bars on the graph represent the standard deviation over the three replicates. The figure shows that for a duration of extraction from 15 min to 1 h, the ultrasonic bath does not allow any improvement of the leach out of MeHg?. The extraction recovery slightly improves with the time, but it never exceeds 45%. The action of the microwave field, on the 8000
1000
MeHg+
6000
cps
750
cps
m/z=202
m/z=202
2+
Hg
500
4000
Hg2+ 2000
250
0
0
0
1
2
t (min)
Fig. 1 Chromatogram of 10 g dm-3 L-Cys
3
4
0
1
2
3
4
t (min)
Fig. 2 Chromatogram of the BCR-463 tuna extract
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Table 2 Certified and experimental mass fractions of BCR-463 Experimental mass fractiona (SD) mg kg-1
Certified mass fractiona mg kg-1
Quantification by the isotope dilution
MeHg?
2.83 ± 0.15
Hg2?
Not given
0.065 (0.002)
Total Hg
2.85 ± 0.16
2.86b (0.08)
2.79 (0.08)
Expressed as Hg
b
Value obtained by summing the species
100
80
60
40
20
0 2 h incubation at 80 °C
15 min US
30 min US
45 min US
60 min US
6 min at 70 W MW
Fig. 3 Extraction efficiency of MeHg? with respect to different protocols (US ultrasonic bath, MW microwave assistance); uncertainties are the standard deviation arising from three replicates
contrary, looks interesting because after only 6 min at 70 W a recovery of about 70% is obtained. A second series of tests therefore focused on the control of extraction with respect to the microwave field (time and power). Two different times were used for different powers (6 or 11 min, equivalent to 1 min for reaching the desired power plus 5 or 10 min of exposition). Results are shown in Fig. 4. The increase of the exposition time allowed an improvement of the extraction efficiency. Moreover, the increase of the power enhances the leach out of MeHg? until quantitative recovery is reached for 140 W. For power above that value, the efficiency decreases, likely due to a process of demethylation of MeHg? during the extraction. Based on these results, the protocol retained for the Fig. 4 Extraction efficiency of MeHg? with respect to the different microwave powers applied and time of exposition; uncertainties are the standard deviation arising from three replicates
The selected protocol was applied with the use of speciesspecific isotope dilution mass spectrometry (SS-IDMS). Any loss of MeHg? occurring during the following sample processing steps does not affect the result thanks to that approach, provided equilibration occurs between all the added isotope enriched MeHg? and the incipient MeHg?. This assumption of equilibration can be assumed if quantitative extraction is reached [15]. As the IDMS procedure is based on a comparison of isotope ratios, which is completely understood, it has the potential to allow traceability of the result to the SI, provided that the isotopically enriched Me200Hg? used as spike has itself values (either certified or measured) for isotopic abundance and mass fraction that are traceable to the SI. Spike of Me200Hg? is therefore calibrated by reverse isotope dilution with a standard of methylmercury with natural abundances checked by the analysis of the CRM IRMM-639 (certified for its isotopic abundances). Purity of MeHg? standard with respect to its mercury species was found to be in the specificity of the producer ([99%) after its analysis by HPLC-ICP-MS. Applying ID quantification procedure, a MeHg? mass fraction equal to (2.87 ± 0.19) mg kg-1 (expressed as Hg) was found in the BCR-463. This result is compatible with the certified value (2.83 ± 0.15) mg kg-1. Thus, the developed analytical approach for the methylmercury determination in fish can be considered as validated. Result uncertainty is indicated as expanded uncertainty (k = 2). Combined standard uncertainty on the results was obtained by propagating together individual uncertainty components according to the ISO/GUM guide [16]. This approach has already been presented in a previous paper [14]. In practice, a dedicated software program was used (Wincert V.3.11.2002.0115, Implex, France). The production of a complete uncertainty budget for the method can itself be seen as an important step in method 100 6 min
11min
80
[MeHg+] extracted (%)
a
[MeHg+] extracted (%)
treatment of samples was the extraction with the microwave assistance at 140 W for 11 min.
60 40 20 0
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70 W
100 W
140 W
210 W
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Repeatability for sample mass fraction determination Repeatability for spike mass fraction determination Repeatability for R in reverse ID Repeatability for R in direct ID
0
5
10
15
20
25
30
35
40
45
50
effect on final uncertainty (%)
Fig. 5 Predominant contributions to the final uncertainty of MeHg? mass fraction; where Cs, Csp are the mass fractions for the sample and the spike, respectively and R the isotope ratios measured during the ID
validation as it allows to highlight the predominant factors affecting the final result. Figure 5 resumes important contributions to the uncertainty of methylmercury mass fraction in the sample. The determination of ratios by the ICP-MS (5 measurement replicates) accounts for more than 70% of the overall uncertainty. Then 20% arrives from the overall method repeatability (4 sample replicates) in which can be included numbers of parameters impossible to estimate individually, e.g. repeatability of sample preparation over the different replicates. In a previous work [14] where an equivalent uncertainty budget was realised for the SS-IDMS of selenomethionine, the same trends were observed for the major factors affecting the overall uncertainty.
Conclusions The use of the HPLC coupled with the ICP-MS has demonstrated its possibilities for the evaluation of mercury species in fish products offering a new alternative to conventional techniques. Moreover, the use of microwaves has confirmed its ability in reducing the time for sample preparation without compromising the determination of methylmercury mass fraction in the sample. Typically, the method presented here requires less than 1 h for sample preparation in only one step before their analysis by HPLC, compared with longer preparation in two steps (extraction then derivatization) for an analysis by GC. Validation of the analytical procedure has been achieved by the analysis of a CRM and the establishment of a complete uncertainty budget. Traceability of measurement
is ensured by the use of double isotope dilution, proper reference materials and also by taking into account every contribution to the overall uncertainty. Therefore, the developed method can be considered as a reference measurement procedure, suitable for the certification of reference materials. At the moment, the use of HPLC-ICP-MS, regarding its sensitivity, is only suitable for matrices with relative high content of mercury. Improvements to decrease its limit of detection are needed for being able to accurately characterise samples with lower mercury concentrations, more representative of natural ones. Nevertheless, the development of alternative procedures is of great interest to improve the assessment of analytes in analytical chemistry, allowing a better evaluation of method performances and impacts on final result.
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