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Direct determination of tin in whole blood and urine by GF AAS
Clinical Biochemistry 46 (2013) 123–127
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Direct determination of tin in whole blood and urine by GF AAS S.V. De Azevedo a,⁎, F.R. Moreira a, 1, R.C. Campos b, 2 a b
Laboratory of Toxicology, Center of Studies of Worker's Health and Human Ecology, National School of Public Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Department of Chemistry, Pontifical Catholic University of Rio de Janeiro (PUC), Brazil
a r t i c l e
i n f o
Article history: Received 29 May 2012 Received in revised form 1 September 2012 Accepted 19 September 2012 Available online 29 September 2012 Keywords: Tin Blood Urine Toxicology Atomic absorption spectrometry
a b s t r a c t Objective: The aim of this work was to develop a procedure for the determination of tin in whole blood and urine by GF AAS with a minimum sample pre-treatment, using Pd/Mg as chemical modifier. Design and methods: The analyses of tin were conducted using an atomic absorption spectrometer with Zeeman background correction. The laboratory staff volunteered blood and urine samples for the experimental studies and application of the methodology. Results: Samples were just diluted with 0.2% v/v Triton X-100, and pyrolysis and atomization temperatures of 1300 and 2200 °C were used. External calibration was performed with matrix matched calibration solutions. Limits of detection of 2.7 and 0.8 μg L−1 were reached for blood and urine, respectively. The method was applied to the determination of Sn in blood and urine of eleven subjects not occupationally exposed, working in a laboratory of toxicology in a large Brazilian city, and the results ranged from 7.4 to 11.2 μg L−1 and ≤0.8 to 2.2 μg L−1, for blood and urine, respectively. Accuracy was assessed by analysis of standard reference materials for tin in blood (Contox I, lot TM144-1097, Kaulson Laboratories, USA) and urine (Seronorm, lot 0511545, Sero AS, Norway). Conclusions: Results showed good agreement between experimental and reference values according to the Student's t test at 95% of confidence. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Tin (Sn) is a naturally occurring metal, obtained from ores such as cassiterite (SnO2). In nature, tin can combine with other elements to form inorganic as well as organotin compounds. Tin and its compounds can be found in air, water and soil, near the rocks, mines and industries where it is present or used [1–3]. Tin is widely used in industry due to features such as low melting point, affinity to form alloys, and corrosion resistance. Inorganic compounds of Sn (iSn) are used in toothpaste, perfume, soap, food additives and dyes [1,4]. The main commercial applications of organic compounds (oSn) are as stabilizers in PVC, pesticides for agricultural use, plastic stabilizers, preservatives (wood, cotton and paper), in the glass industry and as marine antifouling agent [1–3]. Tin metal is used Abbreviations: GF AAS, Graphite furnace atomic absorption spectrometry; AAS, Atomic absorption spectrometry; ICP MS, Inductively Coupled Plasma Mass Spectrometry; Pd, palladium; Mg, magnesium; Pd(NO3)2, palladium nitrate; Mg(NO3)2, magnesium nitrate modifier; Sn, tin; SnO2, tin dioxide; iSn, inorganic compounds of Sn; oSn, organic compounds of Sn; PVC, Polyvinyl Chloride; HNO3, nitric acid; ATSDR, Agency for Toxic Substances and Disease Registry. ⁎ Corresponding author at: Laboratory of Toxicology, Center of Studies of Worker's Health and Human Ecology (CESTEH), National School of Public Health (ENSP), Oswaldo Cruz Foundation (FIOCRUZ), 1480 Leopoldo Bulhões St., Manguinhos, Rio de Janeiro, RJ, CEP 21041-210, Brazil. Fax: +55 21 2270 3219. E-mail address: [email protected] (S.V. De Azevedo). 1 Fax: +55 21 2270 3219. 2 Deceased author.
as a protective coating in food, beverage and aerosol cans. It is also present in alloys such as brass, bronze and pewter, and some welding materials [1,4]. However, it is one of the elements least studied in regard to human health, especially in relation to its presence in biological indicators such as blood and urine [5]. Tin as well as other trace elements occurs naturally in soil at low concentrations but in forms which are not readily available to humans mostly [1,2]. However, some activities such as mining make the metal available to the environment, contributing to the contamination of the surrounding areas [6]. Exposure to Sn and its compounds can produce several effects such as neurological, hematological and immunological. Inhalation of iSn can induce to pneumoconiosis and ingestion may lead to gastrointestinal effects. Exposure to oSn inhibits the synthesis of heme oxygenase and may be genotoxic, while its skin contact may cause severe irritation and burning. Other effects include kidney and liver damage [1,3]. Studies on human health related to tin are still incipient in the literature, in part due to the scarcity of experiments in biological fluids of interest such as blood and urine [5]. Graphite furnace atomic absorption spectrometry (GF AAS) has been widely used for the determination of trace elements in biological fluids due to its low limit of detection, reduced sample volume and minimum sample pre-treatment, saving time and reducing risks of loss or contamination. In addition, other advantageous characteristics such as selectivity, accuracy, precision, and accessibility make the technique very attractive for such determinations [7–13].
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.09.020
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Other sensitive and accurate spectrometric techniques such as ICP MS are turning more popular in the analysis of clinical fluids, but due to its lower costs, especially if a few elements are to be determined, GF AAS is still largely used in the clinical laboratory [10]. Levels of tin in blood and urine reported in the literature are scarce and conflicting [1], and the availability of simple, robust, reliable and accessible procedures may help to change this picture. Thus, the aim of the present study was the development of a sensitive and accurate method for the determination of total tin in blood and urine with a minimum of sample pre-treatment by GF AAS. Experimental Instrumentation A Perkin Elmer (Norwalk, CT., USA) AAnalyst 800 atomic absorption spectrometer equipped with a longitudinal Zeeman-effect background correction system and an AS-800 autosampler was used for all measurements. Integrated absorbance was evaluated exclusively and each measurement was the average of at least three replicates. Instrumental operating parameters of the AAS apparatus are shown in Table 1. Laboratoryware All plastic and glassware were decontaminated by immersion in 5% (v/v) Extran (Merck, Rio de Janeiro, Brazil) overnight and abundantly rinsed with tap water. Afterwards, the material was rinsed with deionized water, immersed in 10% (v/v) HNO3 (Merck) for at least 48 h, copiously rinsed with ultrapure water, obtained from a Milli-Q system (Millipore, Bedford, USA) and dried at 60 °C before use, avoiding any contact with dust and metallic surfaces. Reagents, modifier and standard solutions All the reagents used were of analytical reagent grade. Aqueous standard solutions were daily prepared in 0.2% (v/v) HNO3 (Merck) by appropriate dilutions of a 1000 μg mL −1 Sn standard solution (Merck, New York, USA). Matrix matched calibration curves were prepared using blood and urine samples with Sn content below the limit of detection (LOD), spiked with adequate micro-volumes of the standard solutions and diluted in 0.1% (v/v) Triton X-100 (Merck, Darmstadt, Germany), as described below in Sample preparation section. Solutions of Pd (NO3)2 (Merck, Darmstadt, Germany) and Mg(NO3)2 (Perkin Elmer, Part N°B019-0634), 10 g L−1 each, were diluted in 0.2% (v/v) Table 1 Instrumental operating parameters of the AAS apparatus.
Lamp current Analytical wavelength Background correction system Slit width Mode
Sn Electrodeless Discharge Lamp (EDL) (Perkin Elmer Part n° N3050675) 280 mA 286.3 nm Zeeman effect based (Longitudinal) 0.7 nm Absorbance (peak area)
Graphite furnace operation Atomization tube
Sheath/Purge gas Injection volumes (modifier/sample, μL) Modifier (deposited mass, μg) Diluent (concentration, v/v) Dilution ratio (urine / blood)
Blood and urine samples The laboratory staff volunteered blood and urine samples for the experimental studies and application of the methodology. Eleven whole blood and urine samples were obtained from male and female adults not occupationally exposed and selected randomly. The accuracy of the procedures was checked using reference samples. In the absence of whole blood reference materials, a serum sample was used as control (Contox I Serum, lot TM144-1097, Kaulson Laboratories, USA), with a tin concentration of 3±2 μg L−1. On the other hand, a reference sample of urine (Seronorm Urine, lot 0511545, Sero AS, Norway) at a concentration of 54.6±2.7 μg L−1 was used for controlling the urine. Reference samples were reconstituted according to the manufacturer's instructions and pre-treated as described in Sample preparation section. Sampling and storage Biological samples were collected after the study participants have signed an informed consent form. Whole blood was collected by venipuncture with disposable and sterile needles using vacuum tubes (Vacuette Ltd., SP, Brazil) with heparine as anti-coagulant, specific for the determination of trace elements. Samples of urine were collected in previously decontaminated containers of polyethylene. In addition, subjects were instructed to follow some procedures for proper collection of urine specimens such as washing hands before sampling, not touching inside the lid or container, and close the sampling vessels immediately after collection. Samples were stored at −20 ° C until analysis. Sample preparation Blood and urine samples were diluted in 0.1% (v/v) Triton X-100. The dilution ratios were 1 + 6 and 1 + 4 for blood and urine, respectively. An aliquot of 200 μL of blood or urine was added to 1200 or 800 μL of 0.1% (v/v) Triton X-100, respectively. Calibration curves were daily prepared using four standard intermediate solutions (50, 100, 200 and 500 μg L −1) from a Sn stock 1000 μg L −1 standard in a 0.2% (v/v) HNO3 solution. Then 200 μL of either samples were mixed with 1130 or 750 μL of 0.1% (v/v) Triton X-100 for blood or urine, respectively, and 70 (blood) or 50 (urine) μL of each one of those standard intermediate solutions in a polyethylene tube. After homogenization by mechanical shaking (vortex), an appropriate volume was transferred to the autosampler cup of the GF AAS instrument. All of these operations were performed in a laminar flow hood to avoid contamination. Results and discussion
Operating conditions Primary source
HNO3 in order to obtain 6 μg of Mg(NO3)2 and 10 μg of Pd as Pd(NO3)2 in 10 μL of the modifier solution.
THGA graphite tubes with end caps and integrated platform (Perkin Elmer Part n° B3000655) Argon (Ar) of 99.999% purity (White Martins, Brazil) 10/20 Pd (10) + MgNO3 (6) Triton X-100 (0.1%) 1 + 4/1 + 6
The statistical calculations (uncertainties associated to the calibration curves parameters and comparisons between characteristic mass values and sensitivity ratio to the unit, etc.) were performed according to Gardiner [14], using a 95% confidence level. All the results are an average of at least 3 measurements. The temperature program, type of diluent and dilution ratio as well as modifier mass composition were studied and optimized. These studies were performed by varying the parameter investigated while all others were kept fixed at values shown in Table 1. Temperature program In general, sample and modifier are successively injected before running the temperature program in GF AAS. However, this possibility is not practical in matrices like blood, since subsequent drops of sample and modifier dispensed onto the platform do not mix properly, and
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foaming and ejection are observed during the drying stage, impoverishing the repeatability of the measurements. Moreover, according to the literature [15], protein precipitation may occur when blood sample and modifier solutions are mixed previously to the injection in the furnace. Thus, in the present work, those problems were overcome by injecting and drying the modifier over the platform in the first stage of the temperature program, before addition of the sample solution. Then, blood sample solution was added over a dried modifier layer onto the platform (stage 2). However, since that problem does not occur with the urine, sample and modifier solutions were dispensed conventionally by subsequently pipetting of both solutions and running the temperature program (Table 2). Pyrolysis and atomization temperature curves were performed in the presence of blood and urine matrices as well as with aqueous 50 μg L −1 Sn solutions (in 0.2% v/v HNO3). For these tests, blood and urine samples were diluted 1 + 6 and 1 + 4, respectively, with 0.1% (v/v) Triton X-100, and both were also spiked with 50 μg L −1 of inorganic Sn. In all cases, 10 μL of the modifier solution was used, resulting in 10 μg of Pd plus 6 μg of Mg(NO3)2 deposited onto the platform. The analysis of pyrolysis curves demonstrated that 1300 °C was the best choice as pyrolysis temperature in all situations, expressing the best compromise between maximum sensitivity and minimum background. Accordingly, the background was always within the optimum range correction of the equipment. Regarding the atomization temperature, 2200 ° C appeared as the best one, resulting in maximum sensitivity and a peak profile which could be resolved within only 5 s. Sample dilution One of the impediments to the direct injection of undiluted biological fluids in GF AAS, especially viscous liquids like blood, is the poor repeatability of the dispensed volume generating poor repeatability of the atomic absorption signal. Therefore, sample dilution is necessary, and consequently the choice of suitable diluents (nature and concentration) and dilution factors. In this way, 0.1% (v/v) Triton X-100, water and 0.2% (v/v) nitric acid were studied as diluents, while dilution factors ranging from 3 to 10 for blood and 1.5 to 5 for urine were investigated (Fig. 1). Those choices were based on previous similar studies found in the literature [16]. For these tests, all sample solutions were spiked with 50 μg L−1 of Sn, after the sample dilution. According to Fig. 1, 0.1% v/v Triton X-100 showed the best performance among the diluent solutions studied, leading to largest sensitivities at the lowest dilution ratio. Triton X-100 helps breaking the surface tension, facilitating the sample injection and reducing sample losses to the autosampler capillary walls. Triton X 100 also avoids the clogging of the capillary, which may occur after many readings, especially when dealing with samples such as blood. Clogging obstructs the injection of the correct sample volume onto the platform, leading to inaccurate results. For blood sample, sensitivity losses due to the presence of the matrix were observed up to dilution ratios of 1 + 6, with the signal remaining constant from this ratio. Since no significant sensitivity difference due to the presence of the blood matrix was observed with dilution ratios from 1 + 6 on, this dilution ratio was chosen for the determination of tin in blood, aiming at reaching the lowest Table 2 Temperature program for the determination of tin in blood and urine. Stage
Temperature (°C)
Ramp (s)
Hold (s)
Ar flow (L min−1)
1a 2b 3 4c 5
110 130 1300 2200 2600
10 20 10 0 1
10 30 20 5 3
250 250 250 0 250
a b c
Modifier (blood) or modifier + sample (urine) introduction. Sample introduction (blood). Reading.
Fig. 1. Influence of the diluent nature and dilution ratio in the Sn analytical response in the determination of (a) blood and (b) urine by ET AAS: ( ) 0.1% v/v Triton X-100; ( ) Water; ( ) 0.2% v/v HNO3. Other parameters as in Table 1.
possible limit of detection (LOD) in the original sample. In this way, the background attenuation was well within the correction capacity of the instrument, and no clogging of the autosampler capillary was observed in the long run. Regarding the urine sample, no sensitivity loss due to the presence of the urine matrix was observed in dilution ratios of 1 + 1, 1 + 2 and 1 + 3; however, their use was not advisable since the background attenuation (in peak height) was equal or higher than 1.0. Thus, the dilution ratio of 1 + 4 with 0.1% v/v Triton X 100 was chosen in order to achieve the lowest LOD in the original samples. Modifier A palladium and magnesium nitrate solution was used as chemical modifier, a mixture originally proposed as universal modifier [17]. Performances of three different ratios of Pd+ Mg(NO3)2 were evaluated using 10 μL of these solutions. Masses dispensed onto the platform were 5 + 3; 10 + 6 and 15+ 10 μg of Pd and Mg(NO3)2, respectively. The most appropriate modifier mass composition was assessed by analyzing sensitivities of aqueous and matrix matched calibration curves, with concentrations ranging from 2.5 to 50 μg L −1 of Sn. A significantly lower slope was observed in the presence of lowest modifier masses. However, no significant difference was observed for other masses investigated. Thus, the intermediary modifier mass was chosen. Calibration studies In general, complex matrices like blood and urine have components which interfere in the atomization process, thus preventing the use of aqueous solutions for calibration. In such cases analytical curves prepared in the presence of the matrix may help to eliminate or reduce this interference. Multiplicative matrix effects may be quantified in terms of the sensitivity ratio (m0ac/m0mc), where m0ac is the characteristic mass of the aqueous analytical curve and m0mc
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is the characteristic mass of the matrix matched calibration curve, taken at similar conditions. Multiplicative matrix effects are not present if this ratio does not significantly differ from the unity. In the present case, sensitivity ratios were significantly different from 1 (Table 3) for both matrices, confirming that external calibration cannot be performed with aqueous calibration solutions. Real samples of blood and urine with low tin content were selected and spiked for use. The presence of the blood matrix increased the characteristic mass regarding the aqueous medium in a more usual depressive interference. However, the slopes found in the presence of the urine matrix were higher than those in the aqueous solution, showing an unusual situation. Urine is characterized by a high and variable inorganic salt content such as phosphates, sulfates, chlorides and calcium [4]. According to Borges [18], some of those components can change the Sn behavior in the graphite furnace. The author found that species such as calcium, chloride, and phosphate interfered in the intensity and shape of the analytical signal when determining tin in human milk. Phosphate, which is also present in urine, increased the analytical signal, and it may be the cause of the increased sensitivity in the presence of this matrix. However, analyte addition curves performed in different blood and urine samples at the conditions displayed in Table 1 showed slopes (that is, characteristic masses) with no statistically significant differences (Table 3). Thus, external calibration can be performed using matrix matched calibration solutions, taking samples with the lowest possible Sn content for this matching.
Analytical figures of merit For linearity checking, twenty-one calibration solutions, with concentrations ranging from 2.5 to 500 μg L −1 were used. The aqueous calibration curve was linear up to 150 μg L −1 while linearity was observed up to 50 μg L −1 of Sn in the presence of both matrices. Sensitivity was assessed from the characteristic mass (m0) values, which were calculated from the linear part of the calibration curves. Limits of detection (3 σ, n = 10), calculated [19] for the determination of tin in blood and urine were obtained from the reading of ten different sample solutions of the same sample, with no detectable presence of the metal, after suitable dilution with 0.1% v/v Triton X-100. Values found were 0.8±0.1 and 2.7±0.5 μg L−1 for urine and blood, respectively, in original samples. Limits of quantification [20] (10 σ, n=10) were then 2.5±0.4 and 9.0±1.4 μg L−1 for urine and blood, respectively.
Table 3 Characteristic masses (±SD) and sensitivity ratios in the determination of Sn in blood and urine by GF AAS at optimized conditions. Matrix
Characteristic massa (μg L−1)
Sensitivity ratio (m0a/m0m)b
Blood Average Blood 1 Blood 2 Blood 3 Blood 4 Blood 5 Blood 6 Urine average Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 6
86 ± 5 90 82 91 79 83 90 53 ± 3 56 53 49 56 52 54
0.85 ± 0.03 0.86 0.89 0.82 0.85 0.83 0.87 1.39 ± 0.08 1.30 1.50 1.38 1.31 1.43 1.44
a
Results are averages of six replicates and their standard deviation. m0a/m0m: ratio between the characteristic mass obtained from the calibration curves with aqueous (m0a) and matrix matched standards (m0m) under similar conditions. b
Table 4 Determination of Sn in blood and urine reference materials by proposed procedures at optimized conditions. Sample
Experimental value (μg L−1)
Reference value (μg L−1)
TM144-1097 Seronorm
2.9 ± 1.2 54.3 ± 1.6
3.0 ± 2 54.6 ± 2.7
TM144-1097: Contox Trace Metal Serum Control (Kaulson Laboratories, USA); Seronorm: Seronorm Urine, lot 0511545 (Sero AS, Norway). Results are averages of five replicates and their standard deviation.
The accuracy was assessed by the analysis of standard reference materials. External calibration was performed with matrix matched calibration solutions using blood and urine samples with Sn content below the LOD. Results are shown in Table 4 and there was no statistically significant difference between experimental and reference values. Tin concentrations in blood and urine of environmentally exposed population Blood and urine from eleven volunteers not occupationally exposed to tin have been analyzed using the developed methodology (Table 5). Levels of tin in blood ranged from 7.4 to 11.2 μg L −l while urine concentrations varied from ≤0.8 (LOD) to 2.2 μg L −l. According to the literature [3], the average concentration of tin in blood of nonexposed subjects was approximately 140 μg L −1, (a much larger range than that observed in the present study) while the metal in urine ranged from 0.5 to 5.0 μg L −1, closer to the present data. However, other studies cited by ATSDR reported values ranging from 2 to 9 μg L −1 for blood and 1 to 20 μg L −1 for urine [1]. Many of these studies are outdated, and samples were subjected to processes of pre-treatment such as hot acid digestion requiring a greater number of reagents and steps and they may suffer from analytical uncertainties. Conclusion A simple, sensitive and direct procedure for the determination of Sn in whole blood and urine was developed. Just a dilution step with Triton X100 was necessary as sample pre-treatment as well as the use of a Pd/Mg solution as modifier. Multiplicative matrix effects were observed, impairing the use of aqueous calibration solutions. However, the similarity of the analyte addition curves slopes of a series of samples supported the use of matrix matched calibration solutions, and, in this way, following the optimized temperature program, an agreement statistically significant was observed between experimental and reference values in the analysis of reference materials Also, limits of detection obtained showed that the methodology can be used for the Table 5 Levels of tin in blood and urine (μg L−l) of a not occupationally exposed group. Sample
Blood
Urine
01 02 03 04 05 06 07 08 09 10 11 Mean
8.0 ± 0.4 7.4 ± 0.4 11.2 ± 0.4 10.2 ± 1.8 7.9 ± 0.4 8.8 ± 0.2 7.4 ± 0.4 9.3 ± 0.4 8.7 ± 0.4 7.4 ± 0.4 8.4 ± 0.9 8.4 ± 1.2
≤0.8 ≤0.8 ≤0.8 1.0 ± 0.2 ≤0.8 0.9 ± 0.2 1.0 ± 0.4 ≤0.8 1.6 ± 0.5 2.2 ± 0.7 0.9 ± 0.2 1.0 ± 0.5
Results (μg L−l) are averages of three replicates and their standard deviation.
S.V. De Azevedo et al. / Clinical Biochemistry 46 (2013) 123–127
determination of Sn in blood and urine of people not occupationally exposed. Acknowledgments Authors dedicate this work to the Dr. Campos for his extreme important contribution to this paper. Sayonara Vieira de Azevedo received a scholarship from CAPES. Authors thank CNPq, the Instituto Nacional de Ciência e Tecnologia — Bioanalítica and FIOCRUZ for the financial support.
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Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Direct determination of tin in whole blood and urine by GF AAS S.V. De Azevedo a,⁎, F.R. Moreira a, 1, R.C. Campos b, 2 a b
Laboratory of Toxicology, Center of Studies of Worker's Health and Human Ecology, National School of Public Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Department of Chemistry, Pontifical Catholic University of Rio de Janeiro (PUC), Brazil
a r t i c l e
i n f o
Article history: Received 29 May 2012 Received in revised form 1 September 2012 Accepted 19 September 2012 Available online 29 September 2012 Keywords: Tin Blood Urine Toxicology Atomic absorption spectrometry
a b s t r a c t Objective: The aim of this work was to develop a procedure for the determination of tin in whole blood and urine by GF AAS with a minimum sample pre-treatment, using Pd/Mg as chemical modifier. Design and methods: The analyses of tin were conducted using an atomic absorption spectrometer with Zeeman background correction. The laboratory staff volunteered blood and urine samples for the experimental studies and application of the methodology. Results: Samples were just diluted with 0.2% v/v Triton X-100, and pyrolysis and atomization temperatures of 1300 and 2200 °C were used. External calibration was performed with matrix matched calibration solutions. Limits of detection of 2.7 and 0.8 μg L−1 were reached for blood and urine, respectively. The method was applied to the determination of Sn in blood and urine of eleven subjects not occupationally exposed, working in a laboratory of toxicology in a large Brazilian city, and the results ranged from 7.4 to 11.2 μg L−1 and ≤0.8 to 2.2 μg L−1, for blood and urine, respectively. Accuracy was assessed by analysis of standard reference materials for tin in blood (Contox I, lot TM144-1097, Kaulson Laboratories, USA) and urine (Seronorm, lot 0511545, Sero AS, Norway). Conclusions: Results showed good agreement between experimental and reference values according to the Student's t test at 95% of confidence. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Tin (Sn) is a naturally occurring metal, obtained from ores such as cassiterite (SnO2). In nature, tin can combine with other elements to form inorganic as well as organotin compounds. Tin and its compounds can be found in air, water and soil, near the rocks, mines and industries where it is present or used [1–3]. Tin is widely used in industry due to features such as low melting point, affinity to form alloys, and corrosion resistance. Inorganic compounds of Sn (iSn) are used in toothpaste, perfume, soap, food additives and dyes [1,4]. The main commercial applications of organic compounds (oSn) are as stabilizers in PVC, pesticides for agricultural use, plastic stabilizers, preservatives (wood, cotton and paper), in the glass industry and as marine antifouling agent [1–3]. Tin metal is used Abbreviations: GF AAS, Graphite furnace atomic absorption spectrometry; AAS, Atomic absorption spectrometry; ICP MS, Inductively Coupled Plasma Mass Spectrometry; Pd, palladium; Mg, magnesium; Pd(NO3)2, palladium nitrate; Mg(NO3)2, magnesium nitrate modifier; Sn, tin; SnO2, tin dioxide; iSn, inorganic compounds of Sn; oSn, organic compounds of Sn; PVC, Polyvinyl Chloride; HNO3, nitric acid; ATSDR, Agency for Toxic Substances and Disease Registry. ⁎ Corresponding author at: Laboratory of Toxicology, Center of Studies of Worker's Health and Human Ecology (CESTEH), National School of Public Health (ENSP), Oswaldo Cruz Foundation (FIOCRUZ), 1480 Leopoldo Bulhões St., Manguinhos, Rio de Janeiro, RJ, CEP 21041-210, Brazil. Fax: +55 21 2270 3219. E-mail address: [email protected] (S.V. De Azevedo). 1 Fax: +55 21 2270 3219. 2 Deceased author.
as a protective coating in food, beverage and aerosol cans. It is also present in alloys such as brass, bronze and pewter, and some welding materials [1,4]. However, it is one of the elements least studied in regard to human health, especially in relation to its presence in biological indicators such as blood and urine [5]. Tin as well as other trace elements occurs naturally in soil at low concentrations but in forms which are not readily available to humans mostly [1,2]. However, some activities such as mining make the metal available to the environment, contributing to the contamination of the surrounding areas [6]. Exposure to Sn and its compounds can produce several effects such as neurological, hematological and immunological. Inhalation of iSn can induce to pneumoconiosis and ingestion may lead to gastrointestinal effects. Exposure to oSn inhibits the synthesis of heme oxygenase and may be genotoxic, while its skin contact may cause severe irritation and burning. Other effects include kidney and liver damage [1,3]. Studies on human health related to tin are still incipient in the literature, in part due to the scarcity of experiments in biological fluids of interest such as blood and urine [5]. Graphite furnace atomic absorption spectrometry (GF AAS) has been widely used for the determination of trace elements in biological fluids due to its low limit of detection, reduced sample volume and minimum sample pre-treatment, saving time and reducing risks of loss or contamination. In addition, other advantageous characteristics such as selectivity, accuracy, precision, and accessibility make the technique very attractive for such determinations [7–13].
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.09.020
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Other sensitive and accurate spectrometric techniques such as ICP MS are turning more popular in the analysis of clinical fluids, but due to its lower costs, especially if a few elements are to be determined, GF AAS is still largely used in the clinical laboratory [10]. Levels of tin in blood and urine reported in the literature are scarce and conflicting [1], and the availability of simple, robust, reliable and accessible procedures may help to change this picture. Thus, the aim of the present study was the development of a sensitive and accurate method for the determination of total tin in blood and urine with a minimum of sample pre-treatment by GF AAS. Experimental Instrumentation A Perkin Elmer (Norwalk, CT., USA) AAnalyst 800 atomic absorption spectrometer equipped with a longitudinal Zeeman-effect background correction system and an AS-800 autosampler was used for all measurements. Integrated absorbance was evaluated exclusively and each measurement was the average of at least three replicates. Instrumental operating parameters of the AAS apparatus are shown in Table 1. Laboratoryware All plastic and glassware were decontaminated by immersion in 5% (v/v) Extran (Merck, Rio de Janeiro, Brazil) overnight and abundantly rinsed with tap water. Afterwards, the material was rinsed with deionized water, immersed in 10% (v/v) HNO3 (Merck) for at least 48 h, copiously rinsed with ultrapure water, obtained from a Milli-Q system (Millipore, Bedford, USA) and dried at 60 °C before use, avoiding any contact with dust and metallic surfaces. Reagents, modifier and standard solutions All the reagents used were of analytical reagent grade. Aqueous standard solutions were daily prepared in 0.2% (v/v) HNO3 (Merck) by appropriate dilutions of a 1000 μg mL −1 Sn standard solution (Merck, New York, USA). Matrix matched calibration curves were prepared using blood and urine samples with Sn content below the limit of detection (LOD), spiked with adequate micro-volumes of the standard solutions and diluted in 0.1% (v/v) Triton X-100 (Merck, Darmstadt, Germany), as described below in Sample preparation section. Solutions of Pd (NO3)2 (Merck, Darmstadt, Germany) and Mg(NO3)2 (Perkin Elmer, Part N°B019-0634), 10 g L−1 each, were diluted in 0.2% (v/v) Table 1 Instrumental operating parameters of the AAS apparatus.
Lamp current Analytical wavelength Background correction system Slit width Mode
Sn Electrodeless Discharge Lamp (EDL) (Perkin Elmer Part n° N3050675) 280 mA 286.3 nm Zeeman effect based (Longitudinal) 0.7 nm Absorbance (peak area)
Graphite furnace operation Atomization tube
Sheath/Purge gas Injection volumes (modifier/sample, μL) Modifier (deposited mass, μg) Diluent (concentration, v/v) Dilution ratio (urine / blood)
Blood and urine samples The laboratory staff volunteered blood and urine samples for the experimental studies and application of the methodology. Eleven whole blood and urine samples were obtained from male and female adults not occupationally exposed and selected randomly. The accuracy of the procedures was checked using reference samples. In the absence of whole blood reference materials, a serum sample was used as control (Contox I Serum, lot TM144-1097, Kaulson Laboratories, USA), with a tin concentration of 3±2 μg L−1. On the other hand, a reference sample of urine (Seronorm Urine, lot 0511545, Sero AS, Norway) at a concentration of 54.6±2.7 μg L−1 was used for controlling the urine. Reference samples were reconstituted according to the manufacturer's instructions and pre-treated as described in Sample preparation section. Sampling and storage Biological samples were collected after the study participants have signed an informed consent form. Whole blood was collected by venipuncture with disposable and sterile needles using vacuum tubes (Vacuette Ltd., SP, Brazil) with heparine as anti-coagulant, specific for the determination of trace elements. Samples of urine were collected in previously decontaminated containers of polyethylene. In addition, subjects were instructed to follow some procedures for proper collection of urine specimens such as washing hands before sampling, not touching inside the lid or container, and close the sampling vessels immediately after collection. Samples were stored at −20 ° C until analysis. Sample preparation Blood and urine samples were diluted in 0.1% (v/v) Triton X-100. The dilution ratios were 1 + 6 and 1 + 4 for blood and urine, respectively. An aliquot of 200 μL of blood or urine was added to 1200 or 800 μL of 0.1% (v/v) Triton X-100, respectively. Calibration curves were daily prepared using four standard intermediate solutions (50, 100, 200 and 500 μg L −1) from a Sn stock 1000 μg L −1 standard in a 0.2% (v/v) HNO3 solution. Then 200 μL of either samples were mixed with 1130 or 750 μL of 0.1% (v/v) Triton X-100 for blood or urine, respectively, and 70 (blood) or 50 (urine) μL of each one of those standard intermediate solutions in a polyethylene tube. After homogenization by mechanical shaking (vortex), an appropriate volume was transferred to the autosampler cup of the GF AAS instrument. All of these operations were performed in a laminar flow hood to avoid contamination. Results and discussion
Operating conditions Primary source
HNO3 in order to obtain 6 μg of Mg(NO3)2 and 10 μg of Pd as Pd(NO3)2 in 10 μL of the modifier solution.
THGA graphite tubes with end caps and integrated platform (Perkin Elmer Part n° B3000655) Argon (Ar) of 99.999% purity (White Martins, Brazil) 10/20 Pd (10) + MgNO3 (6) Triton X-100 (0.1%) 1 + 4/1 + 6
The statistical calculations (uncertainties associated to the calibration curves parameters and comparisons between characteristic mass values and sensitivity ratio to the unit, etc.) were performed according to Gardiner [14], using a 95% confidence level. All the results are an average of at least 3 measurements. The temperature program, type of diluent and dilution ratio as well as modifier mass composition were studied and optimized. These studies were performed by varying the parameter investigated while all others were kept fixed at values shown in Table 1. Temperature program In general, sample and modifier are successively injected before running the temperature program in GF AAS. However, this possibility is not practical in matrices like blood, since subsequent drops of sample and modifier dispensed onto the platform do not mix properly, and
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foaming and ejection are observed during the drying stage, impoverishing the repeatability of the measurements. Moreover, according to the literature [15], protein precipitation may occur when blood sample and modifier solutions are mixed previously to the injection in the furnace. Thus, in the present work, those problems were overcome by injecting and drying the modifier over the platform in the first stage of the temperature program, before addition of the sample solution. Then, blood sample solution was added over a dried modifier layer onto the platform (stage 2). However, since that problem does not occur with the urine, sample and modifier solutions were dispensed conventionally by subsequently pipetting of both solutions and running the temperature program (Table 2). Pyrolysis and atomization temperature curves were performed in the presence of blood and urine matrices as well as with aqueous 50 μg L −1 Sn solutions (in 0.2% v/v HNO3). For these tests, blood and urine samples were diluted 1 + 6 and 1 + 4, respectively, with 0.1% (v/v) Triton X-100, and both were also spiked with 50 μg L −1 of inorganic Sn. In all cases, 10 μL of the modifier solution was used, resulting in 10 μg of Pd plus 6 μg of Mg(NO3)2 deposited onto the platform. The analysis of pyrolysis curves demonstrated that 1300 °C was the best choice as pyrolysis temperature in all situations, expressing the best compromise between maximum sensitivity and minimum background. Accordingly, the background was always within the optimum range correction of the equipment. Regarding the atomization temperature, 2200 ° C appeared as the best one, resulting in maximum sensitivity and a peak profile which could be resolved within only 5 s. Sample dilution One of the impediments to the direct injection of undiluted biological fluids in GF AAS, especially viscous liquids like blood, is the poor repeatability of the dispensed volume generating poor repeatability of the atomic absorption signal. Therefore, sample dilution is necessary, and consequently the choice of suitable diluents (nature and concentration) and dilution factors. In this way, 0.1% (v/v) Triton X-100, water and 0.2% (v/v) nitric acid were studied as diluents, while dilution factors ranging from 3 to 10 for blood and 1.5 to 5 for urine were investigated (Fig. 1). Those choices were based on previous similar studies found in the literature [16]. For these tests, all sample solutions were spiked with 50 μg L−1 of Sn, after the sample dilution. According to Fig. 1, 0.1% v/v Triton X-100 showed the best performance among the diluent solutions studied, leading to largest sensitivities at the lowest dilution ratio. Triton X-100 helps breaking the surface tension, facilitating the sample injection and reducing sample losses to the autosampler capillary walls. Triton X 100 also avoids the clogging of the capillary, which may occur after many readings, especially when dealing with samples such as blood. Clogging obstructs the injection of the correct sample volume onto the platform, leading to inaccurate results. For blood sample, sensitivity losses due to the presence of the matrix were observed up to dilution ratios of 1 + 6, with the signal remaining constant from this ratio. Since no significant sensitivity difference due to the presence of the blood matrix was observed with dilution ratios from 1 + 6 on, this dilution ratio was chosen for the determination of tin in blood, aiming at reaching the lowest Table 2 Temperature program for the determination of tin in blood and urine. Stage
Temperature (°C)
Ramp (s)
Hold (s)
Ar flow (L min−1)
1a 2b 3 4c 5
110 130 1300 2200 2600
10 20 10 0 1
10 30 20 5 3
250 250 250 0 250
a b c
Modifier (blood) or modifier + sample (urine) introduction. Sample introduction (blood). Reading.
Fig. 1. Influence of the diluent nature and dilution ratio in the Sn analytical response in the determination of (a) blood and (b) urine by ET AAS: ( ) 0.1% v/v Triton X-100; ( ) Water; ( ) 0.2% v/v HNO3. Other parameters as in Table 1.
possible limit of detection (LOD) in the original sample. In this way, the background attenuation was well within the correction capacity of the instrument, and no clogging of the autosampler capillary was observed in the long run. Regarding the urine sample, no sensitivity loss due to the presence of the urine matrix was observed in dilution ratios of 1 + 1, 1 + 2 and 1 + 3; however, their use was not advisable since the background attenuation (in peak height) was equal or higher than 1.0. Thus, the dilution ratio of 1 + 4 with 0.1% v/v Triton X 100 was chosen in order to achieve the lowest LOD in the original samples. Modifier A palladium and magnesium nitrate solution was used as chemical modifier, a mixture originally proposed as universal modifier [17]. Performances of three different ratios of Pd+ Mg(NO3)2 were evaluated using 10 μL of these solutions. Masses dispensed onto the platform were 5 + 3; 10 + 6 and 15+ 10 μg of Pd and Mg(NO3)2, respectively. The most appropriate modifier mass composition was assessed by analyzing sensitivities of aqueous and matrix matched calibration curves, with concentrations ranging from 2.5 to 50 μg L −1 of Sn. A significantly lower slope was observed in the presence of lowest modifier masses. However, no significant difference was observed for other masses investigated. Thus, the intermediary modifier mass was chosen. Calibration studies In general, complex matrices like blood and urine have components which interfere in the atomization process, thus preventing the use of aqueous solutions for calibration. In such cases analytical curves prepared in the presence of the matrix may help to eliminate or reduce this interference. Multiplicative matrix effects may be quantified in terms of the sensitivity ratio (m0ac/m0mc), where m0ac is the characteristic mass of the aqueous analytical curve and m0mc
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is the characteristic mass of the matrix matched calibration curve, taken at similar conditions. Multiplicative matrix effects are not present if this ratio does not significantly differ from the unity. In the present case, sensitivity ratios were significantly different from 1 (Table 3) for both matrices, confirming that external calibration cannot be performed with aqueous calibration solutions. Real samples of blood and urine with low tin content were selected and spiked for use. The presence of the blood matrix increased the characteristic mass regarding the aqueous medium in a more usual depressive interference. However, the slopes found in the presence of the urine matrix were higher than those in the aqueous solution, showing an unusual situation. Urine is characterized by a high and variable inorganic salt content such as phosphates, sulfates, chlorides and calcium [4]. According to Borges [18], some of those components can change the Sn behavior in the graphite furnace. The author found that species such as calcium, chloride, and phosphate interfered in the intensity and shape of the analytical signal when determining tin in human milk. Phosphate, which is also present in urine, increased the analytical signal, and it may be the cause of the increased sensitivity in the presence of this matrix. However, analyte addition curves performed in different blood and urine samples at the conditions displayed in Table 1 showed slopes (that is, characteristic masses) with no statistically significant differences (Table 3). Thus, external calibration can be performed using matrix matched calibration solutions, taking samples with the lowest possible Sn content for this matching.
Analytical figures of merit For linearity checking, twenty-one calibration solutions, with concentrations ranging from 2.5 to 500 μg L −1 were used. The aqueous calibration curve was linear up to 150 μg L −1 while linearity was observed up to 50 μg L −1 of Sn in the presence of both matrices. Sensitivity was assessed from the characteristic mass (m0) values, which were calculated from the linear part of the calibration curves. Limits of detection (3 σ, n = 10), calculated [19] for the determination of tin in blood and urine were obtained from the reading of ten different sample solutions of the same sample, with no detectable presence of the metal, after suitable dilution with 0.1% v/v Triton X-100. Values found were 0.8±0.1 and 2.7±0.5 μg L−1 for urine and blood, respectively, in original samples. Limits of quantification [20] (10 σ, n=10) were then 2.5±0.4 and 9.0±1.4 μg L−1 for urine and blood, respectively.
Table 3 Characteristic masses (±SD) and sensitivity ratios in the determination of Sn in blood and urine by GF AAS at optimized conditions. Matrix
Characteristic massa (μg L−1)
Sensitivity ratio (m0a/m0m)b
Blood Average Blood 1 Blood 2 Blood 3 Blood 4 Blood 5 Blood 6 Urine average Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 6
86 ± 5 90 82 91 79 83 90 53 ± 3 56 53 49 56 52 54
0.85 ± 0.03 0.86 0.89 0.82 0.85 0.83 0.87 1.39 ± 0.08 1.30 1.50 1.38 1.31 1.43 1.44
a
Results are averages of six replicates and their standard deviation. m0a/m0m: ratio between the characteristic mass obtained from the calibration curves with aqueous (m0a) and matrix matched standards (m0m) under similar conditions. b
Table 4 Determination of Sn in blood and urine reference materials by proposed procedures at optimized conditions. Sample
Experimental value (μg L−1)
Reference value (μg L−1)
TM144-1097 Seronorm
2.9 ± 1.2 54.3 ± 1.6
3.0 ± 2 54.6 ± 2.7
TM144-1097: Contox Trace Metal Serum Control (Kaulson Laboratories, USA); Seronorm: Seronorm Urine, lot 0511545 (Sero AS, Norway). Results are averages of five replicates and their standard deviation.
The accuracy was assessed by the analysis of standard reference materials. External calibration was performed with matrix matched calibration solutions using blood and urine samples with Sn content below the LOD. Results are shown in Table 4 and there was no statistically significant difference between experimental and reference values. Tin concentrations in blood and urine of environmentally exposed population Blood and urine from eleven volunteers not occupationally exposed to tin have been analyzed using the developed methodology (Table 5). Levels of tin in blood ranged from 7.4 to 11.2 μg L −l while urine concentrations varied from ≤0.8 (LOD) to 2.2 μg L −l. According to the literature [3], the average concentration of tin in blood of nonexposed subjects was approximately 140 μg L −1, (a much larger range than that observed in the present study) while the metal in urine ranged from 0.5 to 5.0 μg L −1, closer to the present data. However, other studies cited by ATSDR reported values ranging from 2 to 9 μg L −1 for blood and 1 to 20 μg L −1 for urine [1]. Many of these studies are outdated, and samples were subjected to processes of pre-treatment such as hot acid digestion requiring a greater number of reagents and steps and they may suffer from analytical uncertainties. Conclusion A simple, sensitive and direct procedure for the determination of Sn in whole blood and urine was developed. Just a dilution step with Triton X100 was necessary as sample pre-treatment as well as the use of a Pd/Mg solution as modifier. Multiplicative matrix effects were observed, impairing the use of aqueous calibration solutions. However, the similarity of the analyte addition curves slopes of a series of samples supported the use of matrix matched calibration solutions, and, in this way, following the optimized temperature program, an agreement statistically significant was observed between experimental and reference values in the analysis of reference materials Also, limits of detection obtained showed that the methodology can be used for the Table 5 Levels of tin in blood and urine (μg L−l) of a not occupationally exposed group. Sample
Blood
Urine
01 02 03 04 05 06 07 08 09 10 11 Mean
8.0 ± 0.4 7.4 ± 0.4 11.2 ± 0.4 10.2 ± 1.8 7.9 ± 0.4 8.8 ± 0.2 7.4 ± 0.4 9.3 ± 0.4 8.7 ± 0.4 7.4 ± 0.4 8.4 ± 0.9 8.4 ± 1.2
≤0.8 ≤0.8 ≤0.8 1.0 ± 0.2 ≤0.8 0.9 ± 0.2 1.0 ± 0.4 ≤0.8 1.6 ± 0.5 2.2 ± 0.7 0.9 ± 0.2 1.0 ± 0.5
Results (μg L−l) are averages of three replicates and their standard deviation.
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determination of Sn in blood and urine of people not occupationally exposed. Acknowledgments Authors dedicate this work to the Dr. Campos for his extreme important contribution to this paper. Sayonara Vieira de Azevedo received a scholarship from CAPES. Authors thank CNPq, the Instituto Nacional de Ciência e Tecnologia — Bioanalítica and FIOCRUZ for the financial support.
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