J Radioanal Nucl Chem (2014) 299:241–248 DOI 10.1007/s10967-013-2790-6
Use of neutron activation analysis and LC–ICP-MS in the development of candidate reference materials for As species determination Vivian M. O. Carioni • Cassiana S. Nomura Lee L. Yu • Rolf Zeisler
•
Received: 3 June 2013 / Published online: 7 November 2013 Ó Akade´miai Kiado´, Budapest, Hungary 2013
Abstract Arsenic (As) is widely found in marine organisms, mainly as organic arsenical compounds. With the introduction of regulations for As species in foodstuffs and for environmental control, it has become more important to quantify the amount of arsenic species present. To address this concern new reference materials (RMs) for validation and quality assurance of As speciation measurements are being developed, including a tuna fish tissue and a robalo liver tissue. Instrumental neutron activation analysis (INAA) is used in this work as a proven analytical tool for As determinations and for validating the As mass fraction determined by liquid chromatography coupled to inductively coupled mass spectrometry (LC–ICP-MS) in extracts of the candidate RMs and other fish samples including certified RMs. Various methods for the extraction of watersoluble As species were evaluated. The best results were acquired after methanol/acetone/water extraction yielding in 93 % extractable As in the tuna RM. This procedure was used for the LC–ICP-MS studies. The results demonstrate that INAA can account for 100 % of the distribution of As
V. M. O. Carioni (&) Centro de Ciencias Naturais e Humanas, Universidade Federal do ABC, Santo Andre, SP, Brazil e-mail:
[email protected] V. M. O. Carioni CAPES Foundation, Ministry of Education of Brazil, Brasilia, DF 70040-020, Brazil C. S. Nomura Instituto de Quimica, Universidade de Sao Paulo, Sao Paulo, SP, Brazil L. L. Yu R. Zeisler Chemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, MD, USA
species in analytical processes. It complements LC–ICPMS for the validation of the characterization of As species in the development of RMs for such species. Excellent agreement of measured results in certified RMs with the reported values confirms the validity of the applied procedures. Keywords Arsenobetaine Extraction procedure Reference material Robalo liver Trace elements Tuna tissue
Introduction Arsenic (As), a well-know toxic element and proven human carcinogen, remains a major human health concern. It can occur in high concentrations in products used for human consumption, for example as arsenate of geological origin in some drinking waters. Regulations have set strict limits on this particular source in many countries; however As is abundant in many human foods and almost daily there is news of higher mass fractions of the element being found in food items. This ranges from food staples such as rice, meats, and vegetables to infant formula. As is also widely found in marine organisms, mainly as organic arsenical compounds, since it can be bioaccumulated in marine food chains [1, 2]. Although it is a highly toxic element, its toxicity depends on the chemical form [3] with, for example, one major species, arsenobetaine, found in marine tissues considered nontoxic. Therefore the determination of the total As in a biological sample is not sufficient to evaluate the health and environmental risks and consequently the knowledge of the As species has become important with the introduction of regulations for As content in foodstuffs and for environmental control [3, 4]. This
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concern has led to the development of new analytical methods and to the need for new reference materials (RMs) for validation and control of As speciation measurements [5]. Common procedures for speciation analysis are performed with an extraction of the species followed by separation and identification. In general, As species in seafood can be quantitatively extracted from the samples using solvent extraction methods. Methanol/H2O is commonly used for extraction of polar As species from several biological samples. This mixture has been reported as a highly efficient in the extraction of As from several species of fish, antarctic krill, and shellfish. However, the extraction efficiency of total As seems to be dependent on the matrix composition and the procedure adopted for extraction, ranging between 64 and 100 % [2, 5–9]. Other extraction methods used for As species include methanol/H2O followed by HNO3 extraction for marine periwinkle [10], alkaline alcoholic sample dissolution for fish tissue [11, 12], enzymatic digestion using protonase E and lipase for fish tissues [13] and deionized water for edible algae, fish, and shellfish [9, 14]. The use of apolar solvents such as hexane and acetone for lipids removal prior to As extraction were also reported, and in several cases these solvents were reported to improve the extraction efficiency [2, 5, 15]. These extractions frequently utilize sonication [16], accelerated solvent extraction (ASE) [16], microwaveassisted extraction [11, 14, 17], mechanical agitation [2], and a combination of microwave oven and ultrasonic probe or microwave oven and ultrasonic bath [8]. For separation and determination of the As species liquid chromatography (LC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) has been extensively used and is well established as a method of choice for speciation analysis of several biological samples, including marine organisms [5, 18]. The yield or the amount of As extracted relative to the total As in the sample commonly is reported at 80–90 % depending significantly on type and physical form of the sample. Difficulties have been found when applying these methods for the development of RMs for As speciation determinations, since poor or no information regarding the traceability of the results is presented in the references. For RM development not only should a certified value and its uncertainty be established, but also the metrological traceability should be completely studied since it is fundamental for the usefulness of the RM [19]. According to the international vocabulary of metrology [20], metrological traceability is the ‘‘property of a measurement result whereby the result can be related to a reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty’’. Accordingly, a method is traceable when its measurement data are linked
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to stated references through an unbroken chain of comparisons, all with stated uncertainties [21]. Establishing the traceability of values in RMs, however, is not a trivial task, in particular for speciation analysis, since elemental speciation requires methods performed using many sequential steps including extraction, derivatization, dilution and detection, all susceptible to systematic errors [22]. The aim of this work is to develop reproducible extraction and analytical procedures for the water-soluble As species in candidate RMs leading to traceable quantitative results for total As in the samples as well as in the extracts containing the As species. Instrumental neutron activation analysis (INAA) is suitable for As measurements that meet the metrological requirements [23] and it has been used in a previous study for monitoring extractions of speciation procedures in RMs [24]. Although INAA has been used for direct quantitative determination of As following several modes of separation and collection of As species from water samples [25] the technique is implemented in this work as a tool for validating the amounts of As in As species extracted from a tuna tissue candidate RM and other fish samples including certified RMs (CRMs) and the comparison to As mass fraction in these extracts determined using LC–ICP-MS.
Materials and reagents Test materials Two materials were chosen for the studies: A previously developed tuna fish tissue material ‘‘CMR candidato 1101 Tecido de Atum’’ [26] and a newly developed Robalo liver material. The tuna tissue and the Robalo liver have been acquired from southern Brazil. The preparation of the Robalo liver followed the previously described procedure [26]: the tissue was separated from the viscera, cleaned with deionized water, cut into 2 cm3 pieces, frozen and freeze dried for 48 h. The dried samples were ground using a cutting mill for 3 min at 5,000 rpm and then finely ground using a cryogenic grinder (5 min of freezing followed by 3 cycles of 2 min of grinding, with 1 min of freezing between each cycle). The resulting final product was radiation sterilized, bottled and stored. All tissues were analyzed as received; to report results on a dry mass basis, the moisture content for the materials was evaluated at time of sampling in separate test portions by desiccator drying over MgClO4 using 200 mg of each sample in triplicate. Samples of the CRMs DOLT-3, dogfish liver (National Research Council Canada (NRC), Ottawa, Canada), DORM-3, fish protein (NRC), SRM 1577b, bovine liver (NIST, Gaithersburg, MD, USA), and BCR 627, forms of As in tuna fish (Institute for Reference Materials and
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Measurements, Geel, Belgium) were prepared similar to the RM materials for use as quality control materials. Reagents and chemicals As?3 (AsIII) and As?5 (AsV) standards were purchased from SPEX Certiprep (Metuchen, NJ). The stock solutions of the other individual As species were prepared gravimetrically from dimethylarsinic acid (DMA) (Sigma Aldrich, St Louis, MO, USA), arsenocholine (AC) and arsenobetaine (AB) from Wako Chemicals (Richmond, VA, USA). The liquid chromatography mobile phases were prepared from ACS-grade pyridine and formic acid (Fisher Scientific, Fair Lawn, NJ, USA), and HPLC-grade methanol (JT Baker, Phillipsburg, NJ, USA). The extractions of As species from fish tissue were performed using methanol (JT Baker), acetone (Honeywell Burdick & JacksonÒ, Muskegon, MI, USA), acetonitrile (Alpha AesarÒ, Ward Hill, MA, USA), phosphoric acid (Mallinckrodt Chemical Inc., Paris, KY, USA), TritonÒ X-100 (Acros Organics, Geel, Belgium) hexane and iso-octane (JT Baker). A Continental Water Systems Modulab ModuPure Plus (Sydney, Australia) was used to generate 18 MX cm deionized water used as a solvent in this work.
Procedure Evaluation of the most effective extraction procedure For the evaluation of the As species extraction, 500 mg samples of the candidate RM and CRM materials were used. Initially the efficiency of tuna tissue extraction was evaluated using a methanol/H2O (1:1) solution combined separately with different procedures: (1) agitation for 14 h using 20 mL of the solution; (2) ASE using an accelerated solvent extractor system (ASE 200, Dionex, Sunnyvale, CA, USA) under the following conditions: temperature at 40 °C, pressure at 13.8 MPa, 5 min of equilibration time and 5 min of static time (3 cycles); (3) microwave assisted extraction using a microwave oven system and the following conditions: temperature at 80 °C, 15 min of ramp time, 20 min of hold time and power at 400 W; and (4) ultrasonic bath for 1 h using 20 mL of the extraction solvent. The solutions were centrifuged after each extraction procedure for 10 min. 400 lL of each supernatant were transferred to a filter paper (56 mm diameter Whatman 41 or 542) for posterior INAA. The remaining solid was separated from the supernatant and dried in an oven at 60 °C overnight. Both the solids and the filter paper were pelletized. The supernatants were also ultra-centrifuged for 10 min for 105 gn at 4 °C prior to LC–ICP-MS analysis.
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Evaluation of the most effective solvents The samples were submitted to the ultrasonic bath extraction procedure with different solvents: (a) methanol/H2O 1:1 by volume (20 mL); (b) methanol/Acetone/H2O 1:1:1 (20 mL); (c) H2O with Triton 0.01 % v/v; (d) acetone/H2O 1:1 (20 mL); (e) 10 mL of hexane and 10 mL of H2O; (f) acetone/hexane/H2O 1:1:1; (g) acetonitrile/H2O 1:1 (20 mL); (h) phosphoric acid/acetone/H2O 1:1:1. Determination of element mass fractions by INAA The NIST Research Reactor (NBSR), a 20 MW heavywater cooled and moderated reactor, was the neutron source for INAA in this work. Short (60 s) and long-term (6 h) activation was carried out in the RT-2 pneumatic irradiation channel, which provides a thermal neutron flux of 3.4 9 1013 cm-2/s at a reactor power of 20 MW [27]. The samples were 13 mm diameter 9 1 mm thick pellets of the solid tissues and/or similar size pellets of filter paper onto which solutions of samples or multi-element standards have been pipetted and dried. The samples and standards were sealed into clean linear polyethylene (LPE) pouches for irradiation. After irradiation and appropriate decay samples and standards were transferred to new LPE pouches and sealed for counting. High resolution, high efficiency gamma spectrometers with high purity germanium (HPGe) detectors having relative efficiency of at least 35 % in combination with loss free counting or standard multi-channel analyzers were used [28]. As Species determination by LC–ICP-MS For the determination of As species in the extracts diluted with deionized water (1:100), a LC system (Perkin-Elmer, Shelton, CT, USA) consisting of a Peltier-cooled Series 200 autosampler and a Series 200 quaternary pump equipped with a 200 lL injection loop and a Nucleosil 100-5 SA cation-exchange column (Macherey–Nagel, Bethlehem, PA, USA) was used. A 50 lL injection volume was used for the measurement. The LC conditions used for As species separation was described by Davis et al. [29] and had been previously applied for the separation of As (III), As (V), MMA, DMA, TMAO (trimethylarsine oxide), AB and AC in frozen urine reference materials. The LC system was coupled to a PerkinElmer SCIEX (Thornton, Ontario, Canada) model ELAN DRC II ICP-MS operating in standard mode. The stock solutions of DMA, AB and AC were characterized for use as calibrant. The SI traceability of the calibrants was established using a characterization procedure similar to that previously described [29]. Briefly, a solution containing approximately 14 mg/kg As was prepared by serial dilution from the stock of each species.
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Approximately 0.15 g of the solution was deposited onto 42.3 mm Whatman 41 filter papers. Triplicate filters for each species were prepared for INAA determination of total As in the solution. SRM 3103a As Calibration Standard was used as the calibrant of the INAA measurements. Separately, a dilute sample containing approximately 40 lg/kg As was prepared from the stock solution. Blanks were considered by determining As-containing impurities in the dilute solutions by LC–ICP-MS. The mass fraction of the As species in terms of the As content was calculated by subtracting the As impurity from total As. The associated uncertainty of the measurement was calculated using the procedure described by Duewer et al. [30]. The performance of the LC–ICP-MS method was described by Davis et al. [29].
Results and discussion Total element determination by INAA The first step in this work was the elemental characterization of the candidate RMs by INAA including the analysis of several control materials. Following the established procedures for value assignment in reference materials [31], high quality results for the RM and control materials were obtained; the RM results are presented in Table 1.
Table 1 Results of total element determination in Brazilian candidate RMs by INAA Element
Nuclide, gamma energy (kev)
Al
28
4.97 ± 0.48
7.73 ± 0.29
As
76
5.05 ± 0.14
11.03 ± 0.24
Ca Cl
49
246 ± 50 944 ± 12
737 ± 37 2,059 ± 17
Co
60
Al, 1,778.8 As, 559.08
Ca, 3,084.54 38 Cl, 2167.68 Co, 1,173.24, 1,332.48
Tissue mass fraction (mg/kg)
0.020 ± 0.005
Liver mass fraction (mg/kg)
The accuracy of this INAA procedure was demonstrated using the CRMs DORM-3, DOLT-3, and SRM 1577b, but only for the long irradiation assays because of the special attention placed on As. Table 2 shows these results with the certified values included where available. The measured values are in agreement with the certified mass fractions, although perhaps slightly shifted to about 1 % high, which may be due to the dry mass determination. The total As content in tuna tissue also agrees with results obtained in previous work by HG AAS and SLS-GF AAS [26]. Subsequently this proven INAA procedure was used in all determinations of total As in tissue extracts and residual solids. Extraction procedures for As speciation The choice of a suitable procedure for the quantitative extraction of As from tuna tissue was determined after evaluation of different extraction methods and solvents. INAA was used as the primary tool for the determination of the efficacy of the extraction procedures. Total extractable As was determined in the tissue extracts and the remaining As in the associated residual tissues. These determinations were subsequently corroborated with LC–ICP-MS measurements. The results for different procedures using methanol/H2O are shown in Table 3; the results with different solvents using the ultrasonic bath procedure are shown in Table 4. It should be noted that these initial results are based on peak areas of AB in the chromatograms with mass fractions estimated from a calibration Table 2 Results of total element determination in CRMs DOLT-3, DORM-3 and SRM 1577b by INAA CRM DOLT-3 n = 6
0.38 ± 0.04
Element
Measured value (mg/kg)
As
10.19 ± 0.28
Fe
1,496 ± 79
Certified value (mg/kg) 10.2 ± 0.5 1,484 ± 57
Se
7.23 ± 0.43
7.06 ± 0.48
Zn
91.0 ± 4.9
86.6 ± 2.4
As
6.60 ± 0.21
6.88 ± 0.30
Cr
51
1.20 ± 0.71
0.58 ± 0.08
Cs
134
Cs, 795.87
0.13 ± 0.01
0.030 ± 0.002
Fe
344 ± 26
347 ± 20
Fe
59
Fe, 1,099.25, 1,291.60
42.5 ± 1.5
585 ± 28
3.80 ± 0.27 51.3 ± 2.7
3.3a 51.3 ± 3.1
K
42
Se Zn
6,731 ± 101
3,360 ± 180
Mg Mn
27
Mg, 1,014.43 56 Mn, 846.81
1,237 ± 60 0.19 ± 0.12
682 ± 43 2.86 ± 0.06
Na
24
844.6 ± 8.6
Rb
86
2.67 ± 0.10
1.81 ± 0.16
75
Se
Se
5.42 ± 0.16
3.70 ± 0.17
V
51
Zn
0.034 ± 0.008
0.195 ± 0.015
Zn
65
13.21 ± 0.38
88.5 ± 4.6
Cr, 320.1
K, 1,524.58
Na, 1,368.6 Rb, 1,076.63 Se, 264.66 V, 1,434.2 Zn, 1,115.52
SRM 1577b n = 4
2,450 ± 13
Uncertainties are expanded uncertainties (coverage factor k = 2)
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DORM-3 n = 4
As
\0.06
0.05a
Co
0.260 ± 0.018
0.25a
Fe
187.1 ± 9.4
184 ± 15
Rb
15.05 ± 0.73
13.7 ± 1.1
0.77 ± 0.05
0.73 ± 0.06
127.53 ± 6.1
127 ± 16
Mass fractions values; uncertainties are expanded uncertainties (coverage factor k = 2) a
Noncertified values
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Table 3 Results of As mass fractions in the extracts of tuna tissue using methanol/H2O (1:1) and different extractions procedures Procedure
As extracted (HPLC–ICP-MS) (mg/kg)
As extracted (INAA) (mg/kg)
Remaining As (INAA) (mg/kg)
Agitation
3.64
4.59 ± 0.09
0.58 ± 0.01
ASE
3.24
3.87 ± 0.14
0.69 ± 0.14
Microwave oven
3.6
4.50 ± 0.10
0.56 ± 0.00
Ultrasonic bath
4.16
4.56 ± 0.52
0.52 ± 0.01
Mass fractions values; uncertainties are standard deviation. (Total As in the tuna tissue by INAA measurement = 5.05 mg/kg)
Table 4 Results obtained for total As after extraction of tuna tissue using sonication Method
As extracted (HPLC–ICP-MS) (mg/kg)
As extracted (INAA) (mg/kg)
Remaining As (INAA) (mg/kg)
Methanol/H2O
4.16
4.30 ± 0.8
0.53 ± 0.02
Methanol/Acetone/H20
4.32
4.88 ± 0.27
0.44 ± 0.01
Triton 0.01 % v/v
5.56
4.38 ± 0.12
0.66 ± 0.01
Acetone/H2O
4.11
4.52 ± 0.13
0.50 ± 0.01
a
Hexane/H2O
2.06
3.82 ± 0.16
0.50 ± 0.01
Acetone/Hexane/H2O
2.42
4.12 ± 0.18a
0.48 ± 0.01
Acetonitrile/H2O
3.90
4.49 ± 0.12
0.48 ± 0.01
H3PO4/acetone/H2O
3.12
4.35 ± 0.14
0.62 ± 0.01
Mass fractions values; uncertainties are standard deviation (total As in the tuna tissue by INAA measurement = 5.05 mg/kg) a
In H2O phase
slope established with an AB solution; this approach may be biased but it is sufficient for these determinations. Details on the difference in results between INAA and LC– ICP-MS are discussed below in the section on the determinations of As species by the latter technique. The initial evaluations of different procedures focused on tuna tissue and were conducted with the methanol/H2O solvent described in a number of publications [2, 5–9]. Starting with the mechanically enhanced extractions (agitation, ultra-sound), yields in the 90 % range were achieved. The two additional procedures investigated in this study failed to enhance yields. Microwave oven assisted extraction showed comparable yield, and ASE showed much lower extraction efficiency. The studies were expanded to other solvents to assure that a maximum yield for the extracted As was obtained. According to the literature, yields for a specific method also depend on the species present in the sample, generally high extraction yields (80–90 %) are reported in samples in which AB is the predominant As compounds, as well as in fish tissue [2]. The ultrasonic bath procedure was chosen for the examination of solvents since it is less time
consuming when compared with the overnight agitation and easier to handle, when compared with the microwave oven. Based on the amount of As in the remaining solid fractions, the best results were obtained with the methanol/ acetone/H2O extraction (Table 4), for which 93 % of total As present was contained in the extract. The procedures were further investigated to assure completeness of extraction by repetitive extraction of the tuna tissue RM with the same solvent for three times using the methanol/ H2O and acetone/methanol/H2O solvent mixtures (Table 5). Results for the extracts were obtained by both analytical techniques, showing a decline in the extractable mass fraction for As, as well as for Na and Br by INAA, of more than an order of magnitude for the second extraction and a similar decline was noted with LC–ICP-MS for the third extraction. INAA did not provide reliable results for the second and third extraction because of too high contributions from of the filter paper’s Br background in the gamma-ray spectra causing a high limit of detection for As at the 40 mg/kg level. Based on the LC–ICP-MS results, which showed that the second extraction yielded less than 10 % of the first extraction and the third extraction represented less than 1 % of the total extractable As, it is suspected that the sequential extraction is more a washing effect than an extraction of residual water soluble As species because it is independent of the amounts of water in the solvent mixture. Based on the INAA analysis of the solids, a small amount of the total As (8–12 %) remains in the solid part even after three sequential extractions and washing the solids with pure water. Some As in these tissues is probably bound to proteins, which will not be available to these extractions [32]. Similar results were observed in sequential extractions of the CRMs DORM-3 and DOLT-3. From these studies it was concluded that for quantitative determinations two extractions yield a representative amount of the extractable As. This procedure will be applied to the Table 5 Sequential extractions of tuna tissue using 1:1 volume ratio of methanol:H2O and 1:1:1 volume ratio of acetone:methanol:H2O Step
As extracted As extracted (LC–ICP(INAA) MS) (mg/kg) (mg/kg) Methanol/H2O
As extracted As extracted (LC–ICP(INAA) MS) (mg/kg) (mg/kg) Acetone/methanol/H2O
1
3.60
4.30 ± 1.08
4.32
4.88 ± 0.27
2 3
0.29 0.03
0.63 ± 0.34 0.48 ± 0.42
0.32 0.03
0.80 ± 0.28 0.43 ± 0.08
Total
3.92
4.93 ± 1.21a
4.67
5.47 ± 0.40b
Mass fraction values; uncertainties are standard deviation (n = 3) and combined uncertainties for the sum a
Remaining solid = 0.53 ± 0.02 mg/kg
b
Remaining solid = 0.44 ± 0.01 mg/kg
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determination of AB for value assignment in the tuna tissue RM. Triton and hexane were introduced to better deal with lipids in the tissues but did not increase the amount of As extracted from the tuna tissue as indicated by the amount of As measured in the residual fraction. Measurement of the hexane containing extracts were not quantitative because of the volatile hexane fraction. Consideration of lipids was particularly important with the liver tissue. The initial extractions with methanol/H2O yielded only (4.87 ± 0.12) mg/kg (44 % of total As) water soluble As and (5.44 ± 0.16) mg/kg (49 % of total) with acetone/methanol/H2O. Other extraction procedures, including enzymatic digestion and microwave supported extraction at 80 °C did not change the yield. When hexane was replaced by less volatile iso-octane, (3.51 ± 0.11) mg/kg As (40 % of total As) was extracted in this solvent from the Robalo liver tissue, likely in the form of arsenolipids. Clearly these initial studies point to the need for much more extended investigations on the characterization of the different As species in the liver tissue before quantitative and validated measurements can be undertaken for value assignment of individual species. Determination of As Species by LC–ICP-MS The determinations of As species in the RM tissues were in general agreement with literature values for these types of samples [6]; in all cases AB was the predominant species. The results from the initial determinations were only based on a calibration slope when the extracts from the procedure and solvent performance experiments were analyzed directly after appropriate dilution to the ICP-MS working level. This slope had been established for AB only since other As species did not produce significant peak areas in the chromatograms, perhaps due to the high dilution required for the AB determinations. Therefore differences between LC–ICPMS and INAA are likely due to incomplete and possibly biased quantitative evaluations of the chromatograms. For accurate quantitative determinations the instrumental variations, which lead to suppression of the instrument response, were evaluated. Measurements of six extractions of the tuna tissue RM using acetone/methanol/H2O were performed by adding AC (approximately 50 lg/kg) as an internal standard for the AB determination by LC–ICP-MS. In addition a single point standard addition calibration was used for the determination of AB. The results shown in Table 6 demonstrate an improvement in the precision and accuracy of the AB determination, due to the internal standard compensating for the instrumental drifts. AC was chosen as internal standard because it is not present in the tuna tissue extracts, its elution time is longer than that for AB and it is a molecule more similar to the AB than the
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J Radioanal Nucl Chem (2014) 299:241–248 Table 6 Comparison of AB content in tuna tissue RM after acetone/ methanol/water extraction determined by LC–ICP-MS a) by simple slope calibration and b) after correction using AC as internal standard and calibration with single-point standard addition N
AB (mg/kg), a
AB (mg/kg), b
1
4.58
4.38
2
4.73
4.34
3
4.38
4.37
4
4.79
4.47
5
4.56
4.35
6
4.33
4.37
Average
4.56 ± 0.18
4.38 ± 0.05
other soluble As species considered in this study. As can be seen in Table 6, the corrections can reach 10 % relative. The standard deviation of the results using AC as internal standard and calibration with single-point standard is significantly lower than the standard deviation obtained before the use of the internal standard to correct for instrumental drifts. The effectiveness of the As species extraction and their accurate determination was evaluated with the CRMs DORM-3, DOLT-3 and BCR 627. The yields for soluble As in the extracts were 81.1 % for DORM-3, 87.5 % for DOLT-3, and 89.2 % for BCR 627 as determined by INAA. Considering the sum of the results after INAA determination for As extracted and the remaining As (Table 7), 102 % recovery is calculated for DORM-3 and 99 % recovery is obtained for DOLT-3 and BCR 627. These INAA results can be used to validate the amounts of As determined by LC–ICP-MS in the extracts and show that amounts of extractable As species and amounts of As in the residual tissue sum up to 100 %. Although a small DMA peak could be observed for tissue extractions (Fig. 1), its mass fraction could not be quantified at the estimated level below 0.2–0.8 lg/L [29]. For this reason, AB was the only species evaluated in this work. The CRM BCR 627 has certified values for the As species AB and DMA. INAA showed that the recovery of the total As after extraction equals the total As content in the original tissues. The results by LC–ICP-MS show 100 % recovery of the soluble As species in this analytical procedure; the mass fraction values of (3.99 ± 0.08) mg/kg AB and (0.148 ± 0.01) mg/kg DMA agree with the values in the certificate.
Conclusions Extensive studies of the various approaches for the extraction of water-soluble As species from tissues resulted
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Table 7 Results for CRMs after ultrasonic bath extraction using acetone/methanol/H2O (1:1:1) CRM
As extracted (LC–ICP-MS) (mg/kg)
As extracted (INAA) (mg/kg)
Remaining As (INAA) (mg/kg)
Total As calculated (mg/kg)
Certified value for total As (mg/kg)
DORM-3
5.71 ± 1.01
5.58 ± 0.51
1.44 ± 0.01
7.02 ± 0.51
6.88 ± 0.30
DOLT-3
8.10 ± 0.36
8.93 ± 0.22
1.21 ± 0.18
10.13 ± 0.20
10.2 ± 0.5
BCR 627
4.15 ± 0.10a
4.28 ± 0.18
0.47 ± 0.11
4.75 ± 0.17 4.81 ± 0.11b
4.8 ± 0.3
Mass fractions values; uncertainties are standard deviation for DOLT-3, DORM-3 and BCR 627 (n = 3) a
Sum of AB ? DMA, measured AB = 3.99 ± 0.08; DMA = 0.148 ± 0.010; certified values AB = 3.91 ± 0.23; DMA = 0.15 ± 0.02
b
Measured by INAA
Fig. 1 Chromatogram of the tuna tissue extracted using acetone/methanol/H2O (1:1:1)
in a validated procedure for the extraction from the tuna tissue RM. The ultrasonic bath assisted extraction with methanol/acetone/H2O is efficient and provide excellent yields for extractable As species; it will be used for value assignment of As species in this RM. Quantitative results for the sum of extractable species and residual As by INAA confirmed the reproducibility of the extraction processes. The quantitative determinations of AB with LC–ICP-MS are significantly improved with the implementation of the internal standard method in the latter technique. The combined approach of INAA and LC–ICP-MS has been demonstrated for the selected tuna tissue RM as well as for selected CRMs, however application to other tissues, in particular those with high lipid content, will need additional method development. The results demonstrate that INAA can account for 100 % of the distribution of As species in analytical processes. INAA complements
LC–ICP-MS for the validation of the characterization of As species in the development of reference materials for such species. Excellent agreement of measured results in CRMs with the reported values confirms the validity of the applied procedures. Acknowledgments The authors wish to thank M. M. Schantz, Laura Wood and Siva Chinthalapati of the NIST Chemical Sciences Division for their assistance and valuable suggestions and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, Brazil (FAPESP N° 2007/56504-6), NIST and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior, Brazil (CAPES, N° 0185-12-4) for financial support. Disclaimer Certain commercial equipment, instruments, or materials are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
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