Optimization of different atomic spectrometric methods for the determination of Se in blood and blood fractions

Abstract

ETAAS, FIA-ETAAS and HG-ICP-AES methods were applied for measuring selenium in blood and blood fractions. For ETAAS, the temperature program was optimized, namely the ashing, the pretreatment and the atomization steps. For this purpose real blood samples were used as the matrix. Different chemical modifiers were tested. A combination of Pd and Mg(NO₃)₂ was found to be optimal using a pretreatment temperature of 1100 °C, an ashing temperature of 600 °C and an atomization temperature of 1900 °C. For fractions with low selenium content a multi-fold injection technique was applied. For comparison, measurements by FIA-ETAAS and HG-ICP-AES were performed. They were in good agreement with the results obtained by direct ETAAS measurement. As an application of the method Se was determined in blood and blood fractions (erythrocytes, plasma and lymphocytes) of two groups of people. For the selenium determination in whole blood a detection limit of 0.7 ng ml⁻¹ by ETAAS, of 0.5 ng ml⁻¹ by HG-ICP-AES and of 0.05 ng ml⁻¹ by FIA-ETAAS was obtained.

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Introduction

There is growing interest in the determination of selenium in body fluids because many reports about the beneficial aspects of selenium in diet are being published. Selenium is known to be an essential trace element. It is part of the antioxidative enzyme glutathione peroxidase (GPX), thereby catalyzing the disproportionation of organic peroxides into hydrogen peroxide and molecular oxygen. Se is involved in processes of so-called oxidative stress, which leads to a higher production of free radicals and consequently of peroxides. Therefore, a low selenium status may endanger the defense system against oxidative stress, which is known to be a major contributor to the generation of diseases like cancer, cardiovascular diseases and diabetes mellitus.¹

Since the concentrations of trace elements in blood vary due to changes in human metabolism, their determination in whole blood and blood fractions (plasma, erythrocytes and lymphocytes) is of great importance for gaining information about disorders.^2,3

Usually, only small sample volumes, especially of blood fractions like lymphocytes, are available. Analytical methods have to be chosen, which enable the determination of selenium in blood matrix. A method combining high sensitivity with high reproducibility is advantageous. Atomic spectroscopic methods are frequently used for the determination of selenium. Inductively coupled plasma atomic emission spectrometry (ICP-AES) is known for its high stability and linearity over several orders of magnitude of concentration. Electrothermal atomic absorption spectrometry (ETAAS) is very sensitive and requires only small sample volumes for measurement. Both methods can be hyphenated to a hydride generating system, thereby increasing the sensitivity by one to two orders of magnitude.

In this study primarily ETAAS was used for selenium determination. Since problems are known to exist, causing difficulties for the selenium determination in biological matrices by electrothermal atomic absorption spectrometry, different chemical modifiers are used for the stabilization of selenium.⁴ Using a L'vov platform furnace, atomization is possible under nearly isothermal conditions. Various chemical modifiers have been tested for the selenium determination, the most frequently applied including nickel, palladium and palladium plus magnesium. Different chemical modifiers show different effects for the different species of selenium. For selenium determination in blood plasma Pd plus Mg(NO₃)₂ proved to be optimal.^2,4–9

Ashing of the sample and Zeeman effect background correction are essential for an accurate direct determination of selenium in whole blood and blood fractions.^10–12 An electrodeless discharge lamp increases the sensitivity of determination compared with a hollow cathode lamp because of its higher emission intensity.

Selenium hydride produced by a flow injection analysis (FIA) system can be measured by ETAAS by absorbing the selenium hydride on a graphite furnace coated with iridium.⁹ This method offers lower detection limits because of higher sample volumes used for one replicate. Using a hydride generator for selenium determination¹³ a nearly quantitative analyte introduction is possible, especially for ICP-AES, where the gaseous selenium hydride is introduced into the plasma, thereby improving the sensitivity of the determination compared with liquid sample introduction.¹⁴

The aim of the work was to develop and optimize a fast and reliable method for the determination of selenium for biological samples like blood and blood fractions. The combination of Pd plus Mg(NO₃)₂, known to be optimal for selenium determination in plasma,^15–17 was checked for application to whole blood and erythrocytes. Hydride generating methods (FIA-ETAAS and HG-ICP-AES) were used to prove the results obtained by direct ETAAS measurement.

Due to high individual differences in selenium concentrations in the blood of human subjects it is necessary to analyze many samples from different subjects to obtain statistical significance. The method developed was tested by determining selenium in blood and blood fractions (erythrocytes, plasma and lymphocytes) in two groups different in health status. Fractions (plasma, erythrocytes and lymphocytes) were carefully prepared and the selenium concentrations were measured to enable the localization of possible differences.

Experimental

Instrumentation

For ETAAS measurements a PE 4100 ZL (Perkin Elmer, Norwalk, CT, USA), equipped with a PE FIAS-300 apparatus connected to an AS-90/91 autosampler and an electrodeless discharge lamp (Perkin Elmer), was applied. The operating conditions are described in Table 1a. For details see Experimental section, description of the FIA-ETAAS analysis, and Table 2a and b.

Table 1 (a) Operating conditions for ETAAS; (b) operating conditions for ICP-AES

(a)
Instrument	PE 4100 ZL
Spectrometer	Littrow design
Gratings	1800 lines mm^−1
Atomizing unit	Transverse heated graphite tube
Background correction	Longitudinal Zeeman effect
Magnetic field	0.9 T
Inert gas	Argon
Alternative gas	Air
Autosampler	AS-71
Sample volume	20 µl
(b)
Instrument	ARL 3520 ICP
HF generator	Henry, 27.12 MHz
Rf power supply	1.2 kW
Torch	Fassel type
Ar flow	Outer 12 l min^−1
	Intermediate 0.8 l min^−1
	Inner 1 l min^−1
Sample flow rate	1 ml min^−1
Observation height	15.0 mm
Spectrometer	Paschen–Runge sequential
Gratings	1080 lines mm^−1
Computer	DEC 316x

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Table 2 (a) Operating conditions for FIA-ETAAS, FIA part; (b) operating conditions for FIA-ETAAS, ETAAS part

(a)
FIAS step	Time/s	Pump 1/rpm	Pump 2/rpm	Valve
Prefill	10	100	0	Fill
1	10	100	80	Fill
2	8	0	0	Inject
3	30	0	80	Inject
4	8	0	0	Inject
5	5	0	80	Fill
(b)
ETAAS step	Temperature/°C	Ramp/s	Hold/s	Gas flow/ml min^−1	Gas
1	250	1	15	250	Ar
2	2000	0	5	0
3	2300	1	3	250	Ar

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An ARL 3520 ICP apparatus (ARL, Ecublens, Switzerland) was used for the ICP-AES measurements, coupled to a hydride generator.^13,18 The operating conditions are described in Table 1b.

Reagents

All chemicals used were of reagent grade. Ficoll-Paque^® by Pharmacia Biotech, Uppsala, Sweden; Heparin Immuno^® 5.000 I.E. ml⁻¹ by Immuno AG, Vienna, Austria; nitric acid Suprapur, hydrochloric acid Suprapur, Perhydrol^® (30% H₂O₂) pro Analisi (pA), Triton X 100 pA, Mg(NO₃)₂ pA and single element standard pA by E. Merck, Darmstadt, Germany.

Sample preparation

To obtain samples of whole blood, plasma, lymphocytes and erythrocytes, blood was collected by Venflon to avoid contamination. The first 3 ml were discarded. Heparin was used as an anticoagulating agent (20 µl per 10 ml of whole blood). Erythrocytes were separated from blood plasma by centrifugation (10 min at 1200g) and washed three times using a physiological sodium chloride solution (0.9% m/v). Within 3 h after the blood uptake lymphocytes were separated from diluted blood (1∶1 with physiological sodium chloride solution) by gradient centrifugation (30 min at 1200g) with Ficoll-Paque^®. Lymphocytes appeared as a white ring approximately in the middle of the solution and were collected carefully. They were washed with physiological sodium chloride solution three times and frozen in 1 ml aliquots at −20 °C for further use. To enable comparison of the results the number of erythrocytes and lymphocytes per milliliter were determined by use of a Coulter counter and the concentrations in Tables 3, 4 and 5 are given for the numbers per milliliter listed. For analyses the erythrocytes, plasma and lymphocytes were thawed and homogenized in a vortex mixer. The erythrocyte concentrate was diluted 1 + 9 with doubly distilled water to obtain a hemolyzate. Considering the volume of erythrocyte cells (87%) in the erythrocyte concentrate, the selenium concentrations listed in Table 5 have to be multiplied by a factor of 1.15 to obtain the concentrations for pure erythrocytes, since no selenium is present in the washing solutions prepared with doubly distilled water.

Table 3 Measured concentrations of Se in blood fractions for standard addition compared with direct measurement

	Whole blood/µg l^−1	Plasma/µg l^−1	Erythrocytes/µg l^−1	Lymphocytes/µg l^−1
Direct measurement	69.0 ± 3.4	75.2 ± 3.8	80.7 ± 4.4	0.9 ± 0.4
Standard addition	72.0 ± 3.2	79.6 ± 2.7	83.5 ± 4.5	0.9 ± 0.3

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Table 4 Measured concentrations of Se in blood fractions for comparison of methods

	Blood/µg l^−1	Plasma/µg l^−1	Erythrocytes/µg l^−1	Lymphocytes/µg l^−1
ETAAS	70.2 ± 4.1	63.5 ± 4.2	84.1 ± 5.2	1.3 ± 0.5
FIA-ETAAS	65.2 ± 3.0	—	70.3 ± 3.4	—
HG-ICP-AES	67.5 ± 3.2	—	82.5 ± 2.8	—

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Table 5 Measured concentrations of Se in blood fractions for two collectives of subjects

Group	Blood/µg l^−1	s/µg l^−1	Plasma/µg l^−1	s/µg l^−1	Erythrocytes^a/µg l^−1	s/µg l^−1	Lymphocytes^b/µg l^−1	s/µg l^−1
^aCalculated for 10^10 cells per ml for comparison of results. ^bCalculated for 9 × 10^6 cells per ml.
1 n = 47	68.19	19.26	66.25	18.64	86.30	29.61	1.14	4.81
2 n = 49	71.83	18.11	72.31	17.56	83.11	33.50	0.85	0.51

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Sample preparation for ETAAS. For the ETAAS determination suspensions of the sample in 0.1% nitric acid and 0.1% Triton X 100 were used. For optimization the hemolyzate of the erythrocytes and 1 + 4 dilutions of plasma and whole blood were applied.

Sample preparation for HG-ICP-AES and FIA-ETAAS. Wet digestions have been performed for 12 h at room temperature by adding 5 ml of concentrated HNO₃ plus approximately 5 drops of H₂O₂. Afterwards, the resulting solutions were heated to approximately 120 °C, adding 5 ml of concentrated HNO₃ twice plus approximately 5 drops of H₂O₂. For HG-ICP-AES determination the acidic solutions were reduced by NaBH₄ in a hydride generation system hyphenated to the ICP-AES. For selenium determination the Se^VI initially present in the matrix was reduced to Se^IV by dissolving the residue after the HNO₃–H₂O₂ digestion in 6 M HCl and heating to 80 °C for 20 min.

For both methods standard additions were performed for the selenium determination by adding acidic standard solution to the digested samples (whole blood and erythrocytes).

Optimization of the ETAAS method

Optimization of the temperature program. For erythrocytes and plasma the ashing, pretreatment and atomization temperature of the ETAAS temperature program were optimized (Table 6a). For optimization of the ashing, pretreatment and atomization temperature the following solutions were used: 2000 pg Se standard (0.1% HNO₃); 1000 pg Se + 10 µl matrix (hemolyzate of the erythrocytes, dilutions of whole blood and plasma).

Table 6 (a) Temperature program for the optimization of ETAAS; (b) temperature program for the measurements by ETAAS. Measurement was performed during step 6

	(a)	(b)
Step	T/°C	T/°C	Ramp/s	Hold/s	Gas flow/ml min^−1
Drying	110	110	2	30	250 (Ar)
Drying	130	130	10	40	250 (Ar)
Ashing	400–600	600	10	20	250 (air)
Flushing	600	600	10	10	250 (Ar)
Pre-treatment	700–1500	1100	10	20	250 (Ar)
Atomization	1500–2400	1900	0	5	0
Cleaning	2400	2400	1	2	250 (Ar)

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The temperature curves for optimization were recorded by varying the temperature between 400 and 600 °C for the ashing step, between 700 and 1500 °C for the pretreatment step and between 1500 and 2400 °C for the atomization step. For optimizing the ashing temperature the absorption signal was registered from the ashing step until atomization.

Selection of the modifier. For selenium determination in whole blood as matrix (diluted 1 + 4, 0.1% HNO₃, 0.1% Triton X 100) the following modifiers were used: Cu (20 µg), Mg(NO₃)₂ (3 µg), Ni (20 µg), Pd (5 µg), Ni (10 µg) + Mg(NO₃)₂ (5 µg), Cu (10 µg) + Mg(NO₃)₂ (5 µg) and Pd (5 µg) + Mg(NO₃)₂ (3 µg).

Multi-fold injection. For selenium determination in lymphocytes a volume of 20 µl was injected five times into the graphite furnace, performing drying steps (steps 1 and 2) after each injection.

ETAAS standard addition. Using the autosampler of the ETAAS apparatus, standard additions were performed with the optimized modifier mixture and temperature program for whole blood, plasma, erythrocyte and lymphocyte samples, pipetting different volumes of the standard solution and the sample [(a) 5 + 15 µl; (b) 10 + 10 µl; (c) 15 + 5 µl] into the graphite tube.

Application and comparison of methods

FIA-ETAAS. The graphite furnace was coated by injecting 20 µl of an iridium solution (1000 µg ml⁻¹ in 4 M HCl). The solution was dried and heated to 1200 °C to reduce iridium. For all measurements of whole blood and erythrocytes by FIA-ETAAS 1.2 M HCl was used as carrier solution and 0.2% NaBH₄ in 0.1% NaOH as reducing agent. The sample loop had a volume of 500 µl. For further details see Table 6(a) and (b).

HG-ICP-AES. For HG-ICP-AES the reagent and sample flow were 1.2 ml min⁻¹, and the argon flow 1.1 ml min⁻¹. For reduction of the samples in 6 M HCl, a 0.5% solution of NaBH₄ in 0.1% NaOH and 5% KI was used. For measurement of whole blood and erythrocyte samples the integration time of the emission signal was 5 s. The measuring wavelength was at 196.026 nm.

ETAAS. The conditions optimized previously were used for measurement (listed in Table 6b).

For all methods the detection limit was determined by measuring an appropriate reagent blank solution eleven times and a standard solution three times. The detection limit was calculated as the concentration equivalent to three times the standard deviation of the signal of the blank solution. All standard deviations are based on measurements in triplicate.

Results and discussion

Optimization of the ETAAS method

Optimization of the temperature program. Oxidation temperatures of less than 400 °C were not tested due to fast packing of the graphite tube by blood residues at these temperatures. On increasing the ashing temperature from 400 to 600 °C no significant loss of analyte could be observed. Flushing the graphite furnace with argon before heating to the pretreatment temperature was necessary to avoid oxidation of the graphite tube. For ashing temperatures higher than 600 °C rapid oxidation of the graphite tube was observed. Since the decrease of the absorption signal was less than 2% compared with lower ashing temperatures and a lower background signal was occurring during atomization using an ashing temperature of 600 °C, this temperature was chosen for measurement.

The optimal pretreatment temperature is 1300 °C for standard solutions, according to Fig. 1. In the matrix a significant loss can be observed in all blood fractions using a pretreatment temperature of 1300 °C in spite of the matrix modifier added. In whole blood and erythrocytes sensitivity is decreased by 50% and in plasma by about 30% using a pretreatment temperature of 1200 °C. At 1300 °C, known to be the optimum for pure standard, in all fractions about 80% of the analyte are lost. Therefore, a temperature of 1100 °C was applied, thereby almost avoiding any loss of the analyte during pretreatment (see Fig. 1). In solutions containing blood components a pretreatment temperature of 1100 °C was used, resulting in a higher sensitivity (about twice compared to 1300 °C) and an RSD of about 2%.

Fig. 1 Optimization of the pretreatment temperature in different matrices: erythrocytes ■; standard •; blood ▲; plasma ▼.

For atomization the maximum of the absorption signal was located at about 1900 °C for all matrices of interest (whole blood, plasma and erythrocytes). Since the background and the shape of the peak showed only small changes depending on the atomization temperature, 1900 °C was chosen for measurement.

Selection of the modifier. In our study different matrix modifiers were tested by varying the pretreatment temperature (PT) between 700 and 1400 °;C with an ashing temperature of 600°C and an atomization temperature of 1900 °C.

During pretreatment the matrix should be removed as completely as possible, avoiding any loss of the analyte. Results are shown in Fig. 2. Nickel stabilizes Se compounds using a higher pretreatment temperature, the optimum being 1100 °C. In combination with Mg(NO₃)₂ a better stabilization is reached using a lower pretreatment temperature, but the optimum is still at 1100 °C. Using only copper as modifier, stabilization from 700 to 1200 °C was almost equivalent, but the intensity of the signal was only half compared to the optimal combination. By adding Mg(NO₃)₂ to the copper modifier about two thirds of the maximum intensity was reached, but obviously loss of analyte had occurred already for temperatures higher than 1000 °C. Using only Pd as modifier almost total loss of the analyte during pretreatment was observed: only Mg(NO₃)₂ as modifier guaranteed high sensitivity, although the temperature curve showed decreasing signal intensity for temperatures higher than 1000 °C. A combination of Pd and Mg(NO₃)₂ showed highest sensitivity and almost no changes in intensity on increasing the pretreatment temperature from 700 to 1300 °C. Therefore, according to Fig. 1, the best modifier in whole blood was found to be Pd (5 µg) plus Mg(NO₃)₂ (3 µg), as described in the literature for analysis in plasma.⁴ This combination offers the best sensitivity with a high degree of stabilization of the selenium compounds in whole blood, plasma and erythrocytes during pretreatment.

Fig. 2 Different matrix modifier for selenium determination: Ni ■; Ni+Mg •; Cu+Mg ▲; Pd ▼; Mg ♦; Pd+Mg+; Cu ×.

ETAAS with Zeeman-effect background correction is widely used for the determination of selenium in body fluids, although some selenium species decompose at higher temperatures, resulting in more volatile species leading to loss of analyte. Therefore, chemical modifiers have to be added to stabilize selenium during pretreatment. Quite a number of different chemical modifiers have been proposed, such as copper, nickel and palladium, often in combination with magnesium nitrate, depending on the sample matrix.¹⁷

According to the literature different species of selenium (selenite, selenate, selenomethionine and trimethylselenonium) are not stabilized to the same extent by using chemical modifiers. The species present in blood (selenite, selenate, selenomethionine) are stabilized almost equally by palladium and magnesium nitrate, but not by other modifiers. However, trimethylselenonium is predominantly present in urine but not in blood and blood components. Therefore a mixture of palladium and magnesium nitrate was found to be the best choice for analyses of selenium in plasma.⁴

For all experiments described in the next two sections Pd plus Mg(NO₃)₂ as modifier and the optimized temperature program were used [Table 6(b)].

Multi-fold injection. Multi-fold injection had to be used to allow sensitive measurements with respect to the small concentrations of Se in lymphocyte fractions, although concentration of the analyte was limited to sample injection up to five times because of higher RSD values due to higher matrix load in the graphite tube. Matrix modifier was only added in the first and in the last pipetting step into the tube, otherwise RSDs increased to more than 5%, which was set as the limit for acceptance. Therefore, an injection of 5× 20 µ;l diluted sample (8 parts lymphocyte concentrate + 1 part HNO₃ 1% + 1 part Triton X 100 1%) adding Pd plus Mg(NO₃)₂ modifier in the first and last injection was chosen for measurement. The results are listed in Tables 4 and 5.

ETAAS standard addition. Analyzing 5 samples from one person for each fraction, the results obtained by standard addition were in good agreement with the results obtained by direct measurement (Table 3). Therefore, direct measurement could be applied for the application study.

Comparison of methods

For comparison purposes the FIA-ETAAS method and the alternative HG-ICP-AES method for the determination of the concentration of selenium in whole blood and erythrocytes were applied. The results obtained were in good agreement with the concentrations measured directly by ETAAS (see Table 5). Due to the higher volume necessary for hydride generating techniques ETAAS seems to be advantageous for small sample volumes, as are usually available for lymphocytes. Furthermore, wet digestion, which is necessary for hydride generating techniques, is more time consuming than direct measurement.

For the selenium determination in whole blood a detection limit of 0.7 ng ml⁻¹ for ETAAS, of 0.5 ng ml⁻¹ for HG-ICP-AES and of 0.05 ng ml⁻¹ for FIA-ETAAS, was obtained. ETAAS analysis of selenium in a serum reference material (Seronorm, 94.3 ± 1.5 µg l⁻¹) was in good agreement with the certified value (96 µg l⁻¹, RSD 4.7%).

Application

Results of the measurement in blood and blood fractions of two groups of subjects are listed in Table 5. Using the computer program SPSS 7.5, statistical evaluation was performed by calculating the coefficient of correlation, R, by Pearson. Significance was stated for p < 0.05 or p < 0.01, respectively (p indicates the probability). The relatively high values of the standard deviation in Table 5 are not caused by the determination but by normal physiological deviations.

Concentrations for selenium in erythrocytes are given for 10¹⁰ cells per ml, but actually an average 4–5 × 10⁹ erythrocytes per ml are present in whole blood.¹⁹ Considering the volume of erythrocyte cells (87%) in the suspension investigated, the ratio of the selenium concentrations found inside erythrocyte cells to the selenium concentration found in plasma is 1.5, which is in agreement to the data found in the literature.²⁰ Differences in total selenium in the two groups investigated were found in lymphocytes. The interpretation of correlations of the selenium concentrations with medical parameters and other trace elements will be published elsewhere.

Conclusion

For selenium determination in blood and blood components different methods were chosen for each problem to obtain reliable and precise data.

Hyphenation of a hydride generating system to spectrometric determination (HG-ICP-AES, FIA-ETAAS) was used as alternative method to prove the results obtained by direct ETAAS determination. Using HG-ICP-AES a hydride generator enables a nearly quantitative introduction of the analyte into the plasma, thereby increasing the detection limit compared with fluid sample introduction. Sample, reagent and gas flows have to be optimized carefully for hydride generation.^13,18 HG-ICP-AES enables selenium determination with high sensitivity, usually offering a lower RSD compared with ETAAS, but sample preparation is more time consuming because of the wet digestion necessary and the sample volume needed is about 2 ml, compared with about 100 µl for ETAAS.

FIA combined with ETAAS improves the sensitivity of selenium determination additionally by enabling a preconcentration. Selenium hydride is absorbed on a graphite furnace coated with iridium, thereby enabling the atomization of selenium in up to 1 ml sample volume in one measurement. Similar to HG-ICP-AES wet digestion of the sample is necessary and higher sample volumes are needed for measurement.

Direct measurements by ETAAS enable the determination of low concentrations of selenium in small sample volumes using Pd plus Mg(NO₃)₂ as modifier and the relatively low pretreatment temperature of 1100 °C, although RSDs are higher (about 2%) compared with hydride generation methods (RSDs about 1%). Sample preparation is much faster than for hydride generation methods because no wet digestion is necessary: therefore the direct determination was chosen for the application study. For fractions with a very low selenium content a multi-fold injection technique can be applied successfully, although it is limited to about five injections due to losses during the ashing step and results in higher RSDs (about 5%), which are also due to the low concentration.

Acknowledgements

The authors wish to thank Professor W. Marktl, Institute of Medical Physiology, University of Vienna, Schwarzspanierstrasse 17, A-1090, Vienna, Austria, and Professor G. Schernthaner, Krankenanstalt Rudolfstiftung, Juchgasse 25, A-1030, Vienna, Austria, for their kind support and the medizinisch-wissenschaftlichen Fond des Bürgermeisters der Bundeshauptstadt Wien for financial support.

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