Determination of selenium and tellurium compounds in biological samples by ion chromatography dynamic reaction cell inductively coupled plasma mass spectrometry

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Journal of Chromatography A, 1181 (2008) 60–66

Determination of selenium and tellurium compounds in biological samples by ion chromatography dynamic reaction cell inductively coupled plasma mass spectrometry Chia-Yi Kuo, Shiuh-Jen Jiang ∗ Department of Chemistry, National Sun Yat-sen University, Kaohsiung 80424, Taiwan Received 9 November 2007; received in revised form 13 December 2007; accepted 14 December 2007 Available online 3 January 2008

Abstract An ion chromatography–inductively coupled plasma mass spectrometric (IC–ICP–MS) method for the speciation of selenium and tellurium compounds namely selenite [Se(IV)], selenate [Se(VI)], Se-methylselenocysteine (MeSeCys), selenomethione (SeMet), tellurite [Te(IV)] and tellurate [Te(VI)] is described. Chromatographic separation is performed in gradient elution mode using 0.5 mmol L−1 ammonium citrate in 2% methanol (pH 3.7) and 20 mmol L−1 ammonium citrate in 2% methanol (pH 8.0). The analyses are carried out using dynamic reaction cell (DRC) ICP–MS. The DRC conditions have also been optimized to obtain interference free measurements of 78 Se+ and 80 Se+ which are otherwise interfered by 38 Ar40 Ar+ and 40 Ar40 Ar+ , respectively. The detection limits of the procedure are in the range 0.01–0.03 ng Se mL−1 and 0.01–0.08 ng Te mL−1 , respectively. The accuracy of the method has been verified by comparing the sum of the concentrations of individual species obtained by the present procedure with the total concentration of the elements in two NIST SRMs Whole Milk Powder RM 8435 and Rice Flour SRM 1568a. The selenium and tellurium species are extracted from milk powder and rice flour samples by using Protease XIV at 70 ◦ C on a water bath for 30 min. © 2007 Elsevier B.V. All rights reserved. Keywords: Ion chromatography; Inductively coupled plasma mass spectrometry; Dynamic reaction cell; Speciation analysis; Selenium; Tellurium; Biological samples

1. Introduction Elemental speciation is important in a variety of fields such as environmental, biological, geological and medical applications. The bio-availability, accumulation and toxicological properties of elements are very much dependent on the chemical forms in which they occur in the nature; hence an accurate determination of each species is important to evaluate the potential risk of some elements. The determination of the total element content by most spectroscopic detectors does not provide this often vital information. However, interfacing chromatography with sensitive detection system can provide discrimination of various species. Selenium is well known as an essential element for biological systems but also as a potential toxicant at slightly elevated levels. Tellurium is a non-essential toxic element widely used in metallurgy in the production of steel, cast iron, bronze and also employed as vulcanizing agent, catalyst and in the production of

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0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.12.065

glass [1]. Tellurium concentration is low in real world samples, however, tellurium could be accumulated in milk in which it replaces the essential selenium [2]. Tellurium is usually associated with selenium in minerals and earth crust at trace and ultratrace levels and has similar chemical and physical characteristics [3]. In view of their toxicity, it is necessary to determine various species of selenium and tellurium in environmental samples such as milk powder and rice flour. To understand their metabolism the species are monitored in urine and hence a procedure for speciation of selenium and tellurium is also important in urine samples [4]. As the properties of selenium and tellurium are similar, it is convenient to carry out their speciation simultaneously [5,6]. The inductively coupled plasma mass spectrometer (ICP–MS) is a trace element detection method with unique analytical capabilities. Several reports based on liquid chromatography (LC) [4,7–13], gas chromatography (GC) [12,14] and capillary electrophoresis (CE) [15–19] coupled with ICP–MS for selenium and tellurium speciation analysis have appeared. However, on-line separation methods such as HPLC coupled to ICP–MS gained much attention due to minimal sample pretreatment, losses, contamination and higher sensitivity of ICP–MS.

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The determination of selenium by quadrupole ICP–MS is typically compromised by isobaric interferences from 40 Ar40 Ar+ on 80 Se+ and 40 Ar38 Se+ on 78 Se+ . These interferences are reported to be alleviated by employing dynamic reaction cell (DRC) technique with CH4 as the reaction gas [11,20]. The aims of the present work are to develop a simple extraction procedure and an accurate method of speciation analysis of selenite [Se(IV)], selenate [Se(VI)], Se-methylselenocysteine (MeSeCys), selenomethione (SeMet), tellurite [Te(IV)] and tellurate [Te(VI)] in biological samples using ion chromatography DRC–ICP–MS. The optimization of IC–DRC–ICP–MS operating conditions and its analytical figures of merit, as well as its application to the selenium and tellurium speciation analysis in urines and the extracts of milk powder and rice flour samples are described. 2. Experimental 2.1. IC–ICP–MS device and conditions An ELAN 6100 DRC II ICP–MS (PerkinElmer Sciex, Concord, Canada) was used for these experiments. Samples were introduced by a pneumatic nebulizer with a Scott-type spray chamber. The operating conditions of ICP–MS were optimized by continuous introduction of a solution containing 10 ng mL−1 Se and Te in mobile phase solution. The solution flow rate was maintained at about 1.0 mL min−1 . The ICP–MS operating conditions used in this work are summarized in Table 1. The optimum conditions could vary slightly with time. The HPLC assembly comprised of two HPLC pumps (Hitachi, Model L-6000 & L-6200), an injector (Rheodyne 7255i) and a PRP-X100 anion exchange column (Hamilton, 10 ␮m diameter particles, 250 mm length × 4.1 mm i.d.). Samples were loaded with a syringe into a 200 ␮L sample loop. All separations were performed at room temperature. Each separation was attempted under different combinations of concentrations of ammonium citrate in mobile phase, pH of mobile phase, hold time and ramp time in gradient elution. The conditions listed in Table 1 are those that yielded the best chromatogram of the various sets tested. The column outlet was connected to the pneumatic nebulizer of the ICP–MS through polytetrafluoroethylene (PTFE) tubing. 2.2. Reagents Analytical-reagent grade chemicals were used without further purification. Purified water (18.2 M-cm) from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to prepare all the solutions. Sodium selenite, sodium selenate, sodium tellurite and sodium tellurate were from Alfa (Denvers, MA, USA). Se-methylselenocysteine, seleno-dlmethionine, Protease XIV and ammonium citrate were obtained from Sigma (St. Louis, MO, USA). Ammonium hydroxide was procured from Merck (Darmstadt, Germany). Acetic acid and nitric acid were obtained from Fisher (Fair Lawn, NJ, USA). HPLC grade methanol was from Tedia (Fairfield, OH, USA). Hydrogen peroxide was obtained from Showa (Tokyo, Japan).

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Table 1 Equipment and operating conditions ICP–MS instrument

PerkinElmer Sciex ELAN 6100DRC II

Plasma conditions rf power (w) Plasma gas flow (L min−1 ) Auxiliary gas flow (L min−1 ) Nebulizer gas flow (L min−1 )

1100 15 1.1 1.0

Mass spectrometer settings Resolution Dwell time (ms) Sweeps per reading Readings per replicate Ions monitored Reaction gas Reaction gas flow rate (mL min−1 ) Rejection parameter q Rejection parameter a

0.7 amu at 10% peak maximum 100 10 300 78 Se+ , 80 Se+ , 82 Se+ , 130 Te+ CH4 0.6 0.3 0.0

IC conditions Pump Injector Column

Hitachi, Model L-6000 & L-6200 Rheodyne 7255i Hamilton PRP-X100, 10 ␮m diametre particles, 4.1 mm i.d. × 250 mm length

Mobile phase

A: 0.5 mmol L−1 ammonium citrate in 2% (v/v) CH3 OH (pH 3.7) B: 20 mmol L−1 ammonium citrate in 2% (v/v) CH3 OH (pH 8.0) 0–1 min: 100% mobile phase A 1–1.5 min: 100% A to 100% B 1.5–12 min: 100% mobile phase B After 12 min: 100% mobile phase A

Mobile phase flow rate Sample loop

1.0 mL min−1 200 ␮L

Se(IV), Se(VI), Te(IV) and Te(VI) stock standard solutions (1000 ␮g mL−1 ) were prepared from Na2 SeO3 , Na2 SeO4 , Na2 TeO4 and Na2 H2 TeO6 in pure water, respectively. Working standards were prepared daily from 10 ␮g mL−1 solutions. Se-methylselenocysteine and selenomethionine solutions were stored at −20 ◦ C. 2.3. Sample preparation and extraction The applicability of the developed procedure on the real samples has been carried on two urines, an US National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) RM 8435 Whole Milk Powder, two milk powders obtained locally and a NIST SRM 1568a Rice Flour. Two urine samples collected before taking selenium dietary supplement (Urine 1) and after taking one tablet per day for 7 consecutive days (Urine 2) were analyzed for selenium and tellurium species. These urine samples were diluted 2-folds with pure water. Then a 200 ␮L portion of the sample solution was injected into the IC–ICP–MS system for the determination of selenium and tellurium species. The concentrations of Se and Te species were determined by external calibration method based on peak area. Spike recoveries were measured by spiking the

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diluted urine samples with suitable amounts of selenium and tellurium mixture standard solution. The amounts of selenium and tellurium species present in these sample solutions were quantified by IC–ICP–MS. Recovery was then calculated against the theoretical concentrations. To perform selenium and tellurium speciation analysis of other solid samples, extraction methods adopted must be capable of quantitatively extracting selenium and tellurium from the sample without altering the individual selenium and tellurium species under the extraction conditions. Protease XIV an enzyme used for cell detachment and tissue dissociation, has been used as the reagent for the extraction of various selenium species from biological samples [21,22]. In this work, a simple extraction procedure using Protease XIV as the extracting reagent was employed for the extraction of selenium and tellurium compounds from milk powder and rice flour samples. Approximately 0.5 g each of samples was accurately weighed into 15 mL polyethylene centrifuge tubes and 10 mg of Protease XIV and 10 mL solution of mobile phase B were added. The tube was then put into water bath, maintained at 70 ◦ C, for 30 min. After heating, the samples were allowed to cool and directly centrifuged for 5 min at 3500 rpm. The supernatants were filtered through a PVDF filter (Millipore) of 0.45 ␮m porosity before IC separation. The spike recoveries of individual species were determined by spiking 0.5 g of powder sample with suitable concentration of each of selenium and tellurium mixture standards, dried and then extracted by the extraction solution. The selenium and tellurium standards spiked were 100 ng g−1 each and 20 ng g−1 each in milk powders obtained locally, 40 ng g−1 each and 10 ng g−1 each in NIST whole milk powder, 100 ng g−1 each and 2 ng g−1 each in NIST rice flour, respectively. Since there is no reference value for the real-world samples, the extraction efficiency of selenium and tellurium has been verified by comparing the total selenium and tellurium concentrations in the extracts using the present procedure with those obtained from the complete dissolution method. Samples were digested in a closed microwave oven (MARS 5, CEM) using 3.5 mL HNO3 and 1.5 mL H2 O2 for 0.5 g of milk powder and rice flour [23] and analyzed for total concentrations of Se and Te by DRC ICP–MS using solution nebulization after suitable dilution. The moisture content in rice flour samples has been determined to be 10%. The concentrations obtained were corrected for moisture content for comparison with the certified value of the rice SRM.

nium citrate was selected as the mobile phase in this study [12]. Due to the long retention time of Te(IV) species, gradient elution using ammonium citrate and pH of mobile phase solutions was adopted. The parameters such as concentration of ammonium citrate in mobile phase, pH of mobile phase, hold time and ramp time in gradient elution have been optimized to obtain lower retention times and better resolution. The conditions listed in Table 1 are those that yielded the best chromatogram of the various sets tested. A typical chromatogram of a solution containing 20 ng mL−1 of selenite, selenate, Semethylselenocysteine and selenomethione and 10 ng mL−1 of tellurite and tellurate (as element) is shown in Fig. 1(a). As shown, gradient elution allowed the chromatographic separation of all species in less than 12 min. It may be noted that all the above studies have been carried out in the standard mode of the ICP–MS and ions monitored were 78 Se, 82 Se and 130 Te. The high background at the selenium mass m/z 78 could be due to the 38 Ar40 Ar+ interference which, was reduced in the subsequent experiments using DRC. In fact the background at m/z 80 was too high to record any 80 Se-selective chromatogram in the standard mode. Due to lower sensitivity (Fig. 1a) 82 Se was not considered for further experiments. 3.2. Optimization of DRC conditions As shown in Fig. 1(a), the background at selenium masses m/z 78 and m/z 80 was too high to achieve better detection limit or to record any chromatogram. Quadrupole ICP–MS instru-

3. Results and discussion 3.1. Selection of ion chromatography operating conditions In this study, a conventional pneumatic nebulizer was employed as the sample introduction device. To reduce the organic solvent used, an ion exchange chromatography procedure was employed for the separation of Se and Te species. Several electrolytes were tested as mobile phase for the best separation of selenite, selenate, Se-methylselenocysteine, selenomethione, tellurite and tellurate. After evaluation, ammo-

Fig. 1. Typical mass-selective chromatogram for selenite, selenate, Semethylselenocysteine, selenomethione, tellurite and tellurate: (a) with standard mode, each selenium and tellurium species was present at 20 ng Se mL−1 and 10 ng Te mL−1 , respectively; and (b) with DRC mode. No reaction gas was used in the standard mode. Other HPLC conditions are given in Table 1.

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Fig. 2. Typical chromatogram for the separation of selenium and tellurium species in the (a) diluted urine 1 sample and (b) diluted urine 1 spiked with 5 ng mL−1 each of Se and 0.1 ng mL−1 each of Te mixture standard. The urine was collected before taking selenium dietary supplement. The concentration of Se(IV) and Te (VI) in (a) was about 0.69 and 0.09 ng mL−1 , respectively. Chromatographic conditions used are given in Table 1.

ments equipped with a reaction cell and/or collision cell are known to reduce polyatomic interferences efficiently. The effect of DRC operating conditions on minimizing the spectral interferences arising from 38 Ar40 Ar+ and 40 Ar40 Ar+ that affect the determination of 78 Se+ and 80 Se+ , respectively, were studied carefully. The reaction gas used for this study was CH4 [11,20]. The usual parameters namely the cell gas flow rate and the

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value of rejection parameter q (Rpq) have been optimized to be 0.6 mL min−1 (Ar-equivalent) and 0.3, respectively, that yielded superior signal-to-noise ratio and detection limits. The Rpa value was set at 0 in this study. The rejection parameters Rpq and Rpa are as per the definition reported [24]. By selecting these cell parameters unwanted precursors of interfering species can be filtered out from the ion beam to eliminate interferences created in cell by reaction gas. Moreover, a better estimated detection limit (EDL) could be obtained for 80 Se+ (0.012 ng mL−1 ) compared to 78 Se+ (0.015 ng mL−1 ) and hence, in this study, 80 Se was used for quantification work [20]. In contrast to Se, CH4 gas flow rate did not affect the signal of Te significantly. In fact, there was a slight increase of the Te signal when the CH4 gas flow rate was at 0.5–1.5 mL min−1 . It could be due to the collisional damping which increases the signal of Te. A chromatogram of a solution containing 20 ng Se mL−1 each of Se(IV), Se(VI), SeMeSeCys and SeMet and 10 ng Te mL−1 of Te(IV) and Te(VI) in LC mobile phase is shown in Fig. 1(b). As shown, a significant difference in the background count rates and the detection power was observed between the standard and DRC operating modes. The background at m/z 80 was reduced to less than 50 counts s−1 when ICP–MS was operated under DRC mode. Repeatability was determined using five consecutive injections of a test mixture containing 5 ng mL−1 (as element) each of the selenium and tellurium species studied. From the experiments it was found that the relative standard deviation of the peak areas was less than 5.4% and the repeatability of retention time was better than 1.5% for all species. Calibration curves based on peak area and peak height were linear with correlation coefficients (r2 ) better than 0.9933 for each species in the range studied (0.1–10 ng mL−1 ). The detection limits were estimated from the peak height versus concentration plot and based on the concentration (as element) necessary to yield a net signal equal to three times the standard deviation of the background. The IC–ICP–MS detection limits (as element) were 0.01, 0.03, 0.01, 0.02, 0.08

Table 2 Recoveries and concentrations of selenium and tellurium species in urine samples as measured by IC–ICP–MSa (n = 3) Compound

Urine 1 Spike recovery (%)

TMSe+ ? + MeSeCys SeMet Se(IV) Selenosugar? + Se(VI)

102 103 102 102

± ± ± ±

3b 4 4 4c

Concentration found 22.1 ± 0.8 0.20 ± 0.02 1.38 ± 0.07 25.2 ± 1.2

Spike recovery (%) 98 104 98 105

± ± ± ±

2b 2 2 6c

48.9 ± 1.8 (98%)

Sumd Total

Urine 2 (ng mL−1 )

Te(IV) Te(VI)?

99 ± 3 98 ± 2

n.d.f 0.18 ± 0.01

34.7 ± 1.9 0.45 ± 0.08 1.84 ± 0.03 58.4 ± 1.7 95.4 ± 2.6 (96%)

50.1 ± 1.1

See

Concentration found (ng mL−1 )

98.2 ± 0.6 96 ± 2 106 ± 6

n.d. 0.17 ± 0.01

Sumd

0.18 ± 0.01 (82%)

0.17 ± 0.01 (106%)

Total Tee

0.22 ± 0.02

0.16 ± 0.01

a b c d e f

Values are means of three measurements ± standard deviation. Recovery was determined as described under Section 2. Calculated against MeSeCys. Calculated against Se(VI). The value shown in parenthesis is the percentage of the sum of the species to the total concentration. Determined by pneumatic nebulization DRC ICP–MS. n.d.: not detected.

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and 0.01 ng mL−1 for MeSeCys, SeMet, Se(IV), Se(VI), Te(IV) and Te(VI), respectively. The detection limits obtained in this work are better than or comparable to previous results using similar techniques [6,8,25,26]. 3.3. Sample analysis The proposed procedure was applied for the speciation analysis of selenium and tellurium in the reference materials NIST RM 8435 Whole Milk Powder and NIST SRM 1568a Rice Flour. It was also applied to determine selenium and tellurium species in two milk powder samples purchased from the local market and two urine samples collected in this laboratory. A 200-␮L injection of the diluted urine sample solution was analyzed for Se and Te using the IC–ICP–MS method. Typical mass selective chromatograms (80 Se+ and 130 Te+ ) for urine sample are shown in Fig. 2. As shown, various selenium and tellurium species studied in this work were present in this sample. The assignment of chromatogram peaks was accomplished by analyte addition experiments. The selenium species at approximately 260 and 420 s in urine were identified to be SeMet and Se(IV), respectively, by analyte addition experiments. The species at about 540 s could be selenosugar, which was reported to be the major selenium species in human urine [27]. However, it must be further identified by ESI–MS. There was another unidentified selenium compound near 120 s, it could be trimethylselenium cation [28], selenoadenosylmethionine [29] and/or other organic selenium species [4], however it also needs further identification. From the experimental results, we found that the calibration sensitivities of various selenium species based on peak area were similar. The concentration of unknown was estimated against the average sensitivity of other selenium species studied. The tellurium species at approximately 130 s in urine was identified to be Te(VI) by analyte

Fig. 3. Typical chromatogram for the separation of selenium and tellurium species in the (a) extract of NIST RM 8435 Whole Milk Powder and (b) Whole Milk Powder spiked with Se and Te mixture standard. (b) was obtained by spiking the powder sample with 40 ng g−1 (as element) each of Se and 10 ng g−1 each of Te standard mixture. The concentration of MeSeCys and Te (VI) in (a) was about 0.34 and 0.14 ng mL−1 , respectively. Chromatographic conditions used are given in Table 1.

Table 3 Extraction efficiency of selenium and tellurium species in various samples Sample and element

Concentration (␮g g−1 ) Extracted

RM 8435 Whole Milk Powder Se 0.123 ± 0.002 Te 0.009 ± 0.001

Total

Extraction efficiencya (%)

0.131 ± 0.014b 94 0.010 ± 0.002c 90

Milk powder 1 Se Te

0.639 ± 0.008 0.698 ± 0.016c 92 0.0083 ± 0.0002 0.0090 ± 0.0002c 92

Milk powder 2 Se Te

0.331 ± 0.008 0.361 ± 0.006c 92 0.0097 ± 0.0002 0.0101 ± 0.0006c 95

SRM 1568a Rice Flour Se 0.36 ± 0.01 0.38 ± 0.04b 94 Te 0.0012 ± 0.0001 0.0013 ± 0.0004c 92 Values are means of three measurements ± standard deviation. a The extraction efficiency was calculated on comparison with total concentration. b NIST certified value. c Determined by pneumatic nebulization DRC ICP–MS after sample dissolution.

Fig. 4. Typical chromatogram of (a) extract of NIST SRM 1568a Rice Flour and (b) Rice Flour spiked with Se and Te mixture standard. (b) was obtained by spiking the powder sample with 100 ng g−1 (as element) each of Se and 2 ng g−1 each of Te standard mixture. Chromatographic conditions used are given in Table 1.

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Table 4 Recoveries and concentrations of selenium and tellurium species in NIST standard reference materials as measured by IC–ICP–MSa,b (n = 3) Compound

RM 8435 Whole Milk Powder Spike recovery (%)

Unknown Se MeSeCys SeMet Se(IV) Se(VI)

99 104 100 101

± ± ± ±

2 5 5 3

Concentration found 6.74 3.92 61.5 44.0

± ± ± ±

0.4 0.3 3.8 1.3

Spike recovery (%)

101 ± 5 97 ± 3

3.12 ± 0.65 69.9 ± 1.5 72.8 ± 0.9 108 ± 1 92.6 ± 1.9

105 ± 4 103 ± 3

n.d.d 1.33 ± 0.01

± ± ± ±

346 ± 5 (96%)

5.38 ± 0.18 2.81 ± 0.14 8.19 ± 0.20 (91%)

Sumc

Concentration found (ng g−1 )

1 1 2 3

97 97 103 104

116 ± 4 (94%)

Sumc Te(IV) Te(VI)

SRM 1568a Rice Flour (ng g−1 )

1.33 ± 0.01 (94%)

Values are means of three measurements ± standard deviation. b Recovery was determined by spiking the 0.5 g of NIST RM 8435 Milk Powder and NIST SRM 1568a Rice Flour samples with 5 and 20 ng (as element) each of Te and Se standard mixture and 1 and 50 ng each of Te and Se standard mixture, respectively. c The value shown in parenthesis is the percentage of the sum of the species to the total extracted concentration listed in Table 3. d Not detected. a

Table 5 Recoveries and concentrations of selenium and tellurium species in milk powder as measured by IC–ICP–MSa,b (n = 3) Compound

Milk powder 1 Spike recovery (%)

MeSeCys SeMet Se(IV) Se(VI)

95 96 98 101

± ± ± ±

2 5 6 5

Sumc a b c d

Concentration found (ng g−1 )

Spike recovery (%)

151 ± 3 57.1 ± 1.3 183 ± 2 176 ± 2

100 99 101 99

± ± ± ±

1 4 4 3

567 ± 8 (89%)

Sumc Te(IV) Te(VI)

Milk powder 2

101 ± 2 104 ± 1

n.d.d 7.07 ± 0.12 7.07 ± 0.12 (85%)

Concentration found (ng g−1 ) 75.4 43.4 89.5 92.8

± ± ± ±

0.9 2.6 1.8 0.5

301 ± 4 (90%) 106 ± 5 102 ± 1

3.30 ± 0.34 5.75 ± 0.02 9.05 ± 0.31 (93%)

Values are means of three measurements ± standard deviation. Recovery was determined by spiking the 0.5 g of milk powder with 10 and 50 ng (as element) each of Te and Se standard mixture, respectively. The value shown in parenthesis is the percentage of the sum of the species to the total extracted concentration listed in Table 3. Not detected.

addition experiment, however it could be trimethyltelluronium ion as reported in previous paper [30]. Since a slight change in the retention time and peak width of the elution peaks were observed when various urine samples were injected, peak areas of the elution peaks were used for the quantitative evaluations. The variation of the retention time and peak width of the elution peaks could be due to the difference in the pH or ionic strength of the injected solution. The spike recoveries listed in Table 2 are determined as described in Section 2. As shown, recoveries were in the range of 96–106% for the species studied in different samples. The amounts of Se and Te present in these urine samples were quantified by external calibration method, the results are listed in Table 2. The IC–ICP–MS results were compared with the total concentrations of Se and Te in these urine samples and found to be in satisfactory agreement with the total concentrations (Table 2). In another experiment the urine sample of volunteer, who consumed Se supplement tablets for 1 week, contained elevated concentrations of Se species, trimethylselenium cation and selenosugar in particular. However, Te enrichment has not been occurred in urine samples after Se supplementation.

Extraction of selenium species from biological samples was reported based on enzymatic degradation that result in the recovery of selenomethionine, selenocystein, selenopeptides and selenoproteins [31,32]. Protease is recognized as the most effective enzyme to break the peptide bond down to selenomethionine [32]. Hence, the Se and Te species were extracted from the solid samples using Protease XIV in mobile phase B on a water bath. The total Se and Te concentrations in the extracts were determined and the extraction efficiencies were then calculated by comparing with the total Se and Te values quantified after mineralization. The extraction efficiencies of Se and Te in various samples are given in Table 3. Extraction efficiency was better than 90% for all the samples studied. Typical chromatogram for the separation of selenium and tellurium species in the extract of NIST RM 8435 Whole Milk Powder and NIST SRM 1568a Rice Flour are shown in Figs. 3 and 4, respectively. As shown, various selenium and tellurium species studied in this work were present in the samples. As shown in Fig. 4, there was an unknown peak appeared before MeSeCys peak for rice flour sample. The spike recoveries of individual species have been

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studied by spiking suitable amounts of the species to the samples prior to extraction. The recoveries obtained after IC separations are shown in Table 4. As shown, the recoveries were 97–105% for the species studied in different samples indicating no significant oxidation/reduction and/or decomposition occurred during the extraction process. The amounts of Se and Te present in these samples were quantified by external calibration method and the results are listed in Table 4. The sums of the concentrations of individual species obtained by IC–ICP–MS were in satisfactory agreement with the total concentrations of the elements in the extracts (Table 3). As shown in Table 4, more than 91% of the extractable Se and Te species were present as the species studied in this work. It is interesting to see that compared to milk powder, a larger fraction of selenium in rice was present as organic selenium compounds which are believed to be absorbed by human beings more efficiently. Rice is an important food for the human beings all over the world. The method developed was also applied to determine selenium and tellurium species in milk powder samples obtained locally. However, the tellurium enrichment due to the replacement of selenium cannot be commented as the analysis has been carried out on a commercial milk powder sample. The results are listed in Table 5. 4. Conclusion The merits of coupling ion chromatography and ICP–MS with conventional pneumatic nebulization for selenium and tellurium speciation analysis in biological samples have been demonstrated. The concentrations of various selenium and tellurium compounds in urine, milk powder and rice flour have been determined. The utility of DRC–ICP–MS in alleviating the interferences during the determination of Se has also been shown. A simple extraction procedure by using Protease XIV at 70 ◦ C on water bath yielded near quantitative recoveries of all the species without any oxidation/reduction and/or decomposition. The developed procedure can be utilized for the routine monitoring of these species in biological samples as it is simple, rapid and easy to adopt. Acknowledgement This research was supported by a grant from the National Science Council of the Republic of China under Contract NSC 95-2113-M-110-007. References [1] P. Cava-Montesinos, M.L. Cervera, A. Pastor, M. Guardia, Anal. Chim. Acta 481 (2003) 291.

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