Selenium determination in biological fluids using Zeeman background correction electrothermal atomic absorption spectrometry

Procedures for the direct determination of total selenium in urine, serum, and blood using electrothermal atomic absorption spectrometry are presented. In the selected experimental conditions, Zeeman correction is mandatory to compensate for the high background signals. The sample diluted and containing 0.1% (w/v) Triton X-100 is introduced directly into the electrothermal atomizer. A solution containing 15% (w/v) hydrogen peroxide, 0.65% (w/v) nitric acid, and 0.5% (w/v) nickel is injected separately into the atomizer. Calibration is carried out using the standard additions method. The detection limit is 30 pg selenium. If palladium, instead of nickel, is used as the chemical modifier, calibration can be carried out against aqueous standards, and the detection limit is 45 pg. In this case, three separate injections are required to prevent precipitation problems in the automatic injector. The reliability of the procedures is checked by analyzing three certified reference materials and by recovery studies. Mean recoveries are 99.7% for serum, 99.4% for urine, and 100.8% for blood samples. Relative standard deviation values are +/-4.0% for serum, +/-3.9% for urine, and +/-4.5% for blood.     

Direct determination of selenium in rat blood plasma by Zeeman atomic absorption spectrometry

The method was developed to be applied for direct determination of selenium in rat plasma by graphite-furnace atomic absorption spectrometry with Zeeman background correction. Blood was obtained from CD rats of both sexes 2h after dosing in weeks 7 and 13 in order to acquire data on the levels of selenium in these animals during 13-week gavage administration of l-seleno-methylselenocysteine (SeMC), a new candidate chemopreventive agent under development. Application of the commonly used method of standard addition was found to be unsuitable to calculate the selenium content in rat plasma (within-run and between-run accuracy and precision parameters were less than 85%). Therefore, a new analytical method was developed. In this method, samples of rat plasma (50 microL) were diluted 10-fold with a reducing agent containing l-ascorbic acid, a modifier solution containing palladium chloride and Triton X-100. Samples were atomized in pyrolytically coated graphite tubes and peak height signals were measured. Selenium concentrations were determined by linear least squares regression analysis based on the standard curve generated in pooled rat blank plasma. Since selenium is normally present in plasma, a three-step approach was used to calculate selenium plasma levels. Initially selenium levels were determined based on the standard curve with selenium-spiked pool plasma. In the second step, background selenium levels in the pooled plasma were determined based on the same standard curve. In the third step, background level was added to the previously derived number. The relative errors were in the range from -4.6 to 11.4% (intra-day assay) and from -0.4 to 8.8% (inter-day assay) which proved good accuracy. The relative standard deviations were in the range from 1.88 to 4.70% (intra-day precision) and from 3.28 to 5.38% (inter-day precision). In rat plasma, the following dose-dependent selenium levels (mean+/-S.D.) in males and females, respectively, were observed at 13 weeks: 655.5+/-48.8 and 595.8+/-43.9 ng/mL (control group), 927.9+/-85.3 and 859.3+/-164.3 ng/mL (0.4 mg/kg per day dose group), 1238.9+/-182.4 and 1169.9+/-112.6 ng/mL (0.8 mg/kg per day dose group), and 1476.5+/-138.1 and 1320.1+/-228.6 ng/mL (2.0mg/kg per day dose group). No significant sex differences in selenium plasma levels were seen in the SeMC-treated groups. No significant differences in selenium plasma levels were seen between mean plasma levels at 7 and 13 weeks. The described method is simple, rapid, accurate, precise and can be easily applied in other laboratories for a large number of samples.     

Comparison of the serum selenium content of healthy adults living in the Antwerp region (Belgium) with recent literature data.

Graphite furnace atomic absorption spectrometry, after improved matrix modification and using Zeeman background correction, was used to measure the serum selenium content of healthy adults living in the Antwerp region (Belgium). The mean serum concentration of 13 men and 13 women, sampled once a month during 1 year, was 84.3 +/- 9.4ng/ml with a broad range of 51.4-121.7 ng/ml. The intra-individual variation was remarkably high. Recent literature on selenium concentrations is reviewed and values are tabulated, with limitation to healthy adults and European countries. The mean serum selenium concentration measured corresponded well to older literature data for Belgium. The obtained values were found to be in the medium range compared with the literature data for other European countries.     

Plasma selenium levels in healthy blood bank donors in the central-eastern part of Belgium

Graphite furnace atomic absorption spectrometry, with Zeeman background correction and after improved matrix modification, was used to measure the plasma selenium content of healthy blood bank donors in the central part of Belgium.

The mean plasma selenium concentration of 80 men and 80 women was 79.7±4.4 ng/mL with a range of 55.0–117.4 ng/mL.

There was no gender difference observed. Plasma selenium level was significantly highest for the adult group, aged 45–64 years, compared to the others, except the young adults (18–24 years).

The mean plasma selenium concentration measured corresponded well with literature data for Belgium. The obtained values were found to be in the medium range, compared with recent literature values for the European countries.     

Plasma selenium status in a group of Australian blood donors and fresh blood components

The purpose of this study was to assess plasma selenium levels in an Australian blood donor population and measure extra-cellular selenium levels in fresh manufactured blood components. Selenium levels were measured using graphite furnace atomic absorption spectrometry with Zeeman background correction. The mean plasma selenium level in healthy plasmapharesis donors was 85.6 ± 0.5 μg/L and a regional difference was observed between donors in South East Queensland and Far North Queensland. Although participants had selenium levels within the normal range (55.3–110.5 μg/L), 88.5% had levels below 100 μg/L, a level that has been associated with sub-optimal activity of the antioxidant enzyme glutathione peroxidase (GPx). Extra-cellular selenium levels in clinical fresh frozen plasma (cFFP) and apheresis-derived platelets (APH Plt) were within the normal range. Packed red blood cells (PRBC) and pooled buffy coat-derived platelets (BC Plt) had levels at the lower limit of detection, which may have clinical implications to the massively transfused patient.     

Atomic energy of Canada Limited

A method is described for determining stable cobalt concentrations in natural lake waters using polarized Zeeman effect graphite furnace atomic absorption spectrometry. The analysis requires small sample volumes, minimal sample pretreatment and preparation and no chelation or solvent extraction procedures. Instrument linearity, per cent cobalt recovery, matrix interferences and the use of the standard additions method are discussed. The increased sensitivity and precision due to Zeeman AA background correction permits precise determination of stable cobalt in the µg.1^–1 range.AECL 8351 reprintAECL 8351 reprint     

Determination of selenium via the fluorescence quenching effect of selenium on hemoglobin‐catalyzed peroxidative reaction

A new method for the determination of selenium based on its fluorescence quenching on the hemoglobin‐catalyzed reaction of H₂O₂ and l ‐tyrosine has been established. The effect of pH, foreign ions and the optimization of variables on the determination of selenium was examined. The calibration curve was found to be linear between the fluorescence quenching (F₀/F) and the concentration of selenium within the range of 0.16‐4.00 µg/mL. The detection limit was 1.96 ng/mL and the relative standard deviation was 3.14%. This method can be used for the determination of selenium in Se‐enriched garlic bulbs with satisfactory results. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of selenium stable isotopes by gas chromatography–mass spectrometry with negative chemical ionisation

A gas chromatography mass spectrometric method using negative chemical ionisation was developed for the determination of stable isotopes of selenium for evaluation of selenium absorption and retention from foods in humans. The method involves an acid digestion to convert all selenium into selenite, which subsequently reacts with 4-nitro-o-phenylene-diamine to form a volatile piazselenole. The piazselenole, after extraction into an organic solvent, was analysed for its isotopic selenium composition by gas chromatography mass spectrometry. Negative chemical ionisation is reported for the first time for the determination of selenium stable isotopes and its analytical characteristics were compared to those of electron impact mass spectrometric ionisation, classically used for the determination of selenium. The negative chemical ionisation technique allowed accurate determination of total selenium by isotope dilution and of selenium isotope ratios in biological samples. The repeatability for total selenium and for stable isotope ratios was good (R.S.D.≤10%) within the range of 50 to 250 ng selenium. The detection limit for the investigated selenium isotopes was approximately 1 pg (signal to noise ratio at 3). The applicability of the developed stable isotope methodology was demonstrated by the determination of the selenium absorption and retention from foods in a pilot study using one human adult.     

Determination of selenium in serum by FI-HG-AAS and calculation of dietary intake

A method was developed for the determination of selenium concentration in serum by flow injection-hydride generation-atomic absorption spectrometry (FI-HG-AAS) following microwave digestion of serum samples and reduction of selenate to selenite. The detection limit of the method was 0.3 μg Se/L and the characteristic concentration, corresponding to the 0.0044 absorbance signal, was 0.12 μg Se/L. The results from the analysis of two Seronorm standard reference materials showed good agreement with the certified values. The method was then used to analyze selenium in sera of Austrian and Slovenian people for the calculation of dietary intakes. The selenium concentrations in sera of mothers at delivery, their neonates, and the male and female adults were 71 ± 14, 42 ± 6, 75 ± 21, and 65 ± 16 μg/L for the Austrians and 62 ± 15, 34 ± 7, 70 ± 12, and 66 ± 15 μg/L for the Slovenians. The dietary intakes of selenium of the mothers and the male and the female adults were calculated as 52, 37, and 46 μg/d for the Austrians and 45, 38, and 32 μg/d for the Slovenians.     

Microdetermination of selenium in protein fractions isolated by analytical methods.

A method has been developed for the determination of selenium associated with proteins in chromatographic fractions, in polyacrylamide gels, and on nitrocellulose membranes after transfer. This method involves the complete digestion of samples in the purest nitric and perchloric acids and takes advantage of the specificity afforded by the 99% pure 2,3-diaminonaphthalene. The use of these and other reagents of highest purity produces very low blank values and allows a detection limit as low as 0.76 picomoles (60 picograms) of selenium. Quantitative recoveries of selenium in glutathione peroxidase and low coefficients of variation were obtained.     

Microdetermination of selenium in protein fractions isolated by analytical methods

A method has been developed for the determination of selenium associated with proteins in chromatographic fractions, in polyacrylamide gels, and on nitrocellulose membranes after transfer. This method involves the complete digestion of samples in the purest nitric and perchloric acids and takes advantage of the specificity afforded by the 99% pure 2,3-diaminonaphthalene. The use of these and other reagents of highest purity produces very low blank values and allows a detection limit as low as 0.76 picomoles (60 picograms) of selenium. Quantitative recoveries of selenium in glutathione peroxidase and low coefficients of variation were obtained.     

Whole Blood Thallium Determination by GFAAS with High-Frequency Modulation Polarization Zeeman Effect Background Correction

A new technique of blood thallium direct determination based on graphite furnace atomic absorption spectrometry with Zeeman background absorption correction system was designed. The developed technique does not require sample digestion. Sample treatment includes only a fivefold per volume dilution of blood sample with 0.1% (m/v) Triton X-100. L’vov integrated platform was modified with 400 μg of Rh. Matrix modifier (200 μg NH₄NO₃ and 160 μg Pd(NO₃)₂) was suggested for coping chloride and blood organic matter interferences. Standard reference material (Clincheck® Plasma Control for trace elements) analysis was used for validation. Additional validation was performed by analyzing spiked blood samples in the whole dynamic range. The dynamic range was 2–50 μg/L. Precision (RSD) was found <12%. Blood thallium limit of detection was 0.2 μg/L.     

Rapid and direct determination of selenium, copper, and zinc in blood plasma by flow injection-inductively coupled plasma-mass spectrometry

A flow injection-inductively coupled plasma-mass spectrometry (FI-ICP-MS) with a simple sample preparation procedure was developed for the determination of selenium, copper, and zinc in blood serum/plasma. A serum/plasma sample was filtered through a 0.45-μm membrane filter and diluted with a mixture of trace elements in a standard solution (9∶1, v/v). Measurement of the reference serum sample confirmed the accuracy of our method for selenium, copper, and zinc concentration. In the case of blood plasma samples obtained from six healthy adult males, the selenium, copper, and zinc concentrations were similar to those of a typical healthy male in Japan. These results suggest that the sample prepartive procedure coupled with FI-ICP-MS can be used for the routine determination of selenium, copper, and zinc in human blood serum/plasma.     

Improved method for selenium determination in biological samples by gas chromatography

A gas chromatographic assay employing electron-capture detection for the determination of selenium in biological samples is reported. A calibration curve of 4-nitro-o-phenylenediamine derivative of selenium as a function of peak area was linear from 5–1000 pg. The limit of detection for the electron-capture detector was approximately 0.5 pg. Recoveries of selenium added to various biological materials ranged from 95–105%. This procedure reduces the number of transfers thereby reducing errors associated with losses or contamination. One advantage of the present method is that interfering compounds occurring in previously employed chromatographic methods are eliminated. This procedure can be used for routine microanalysis of selenium. Samples containing less than 2 ng selenium in 200 μl of biological fluid can be routinely analyzed using this method.     

Sensitive determination method of estradiol in plasma using high-performance liquid chromatography with electrochemical detection

The improvement in the sensitive determination method of estradiol using HPLC with electrochemical detection is described. The improvement was due to the optimization of the potential applied to the electrode of the analytical cell and employment of a guard cell. The detection conditions were optimized from the electrochemical properties of estradiol in acidic and alkaline eluents. The employment of the guard cell drastically decreased the background noise without any reduction in the response of estradiol, and contributed to improvement in the sensitivity. The optimized method combined with pretreatment by liquid-liquid extraction was applied to the determination of estradiol in rat plasma. The detection limit of 8 pg for the standard solution and 24 pg for the plasma sample, which was about 6-8-fold more sensitive compared to the previous reports, was attained.     

Principles and methods for the quantitative determination of feulgen stained DNA with the television texture analysis system (TAS)

Summary The television texture analysis system (TAS, Leitz) has been applied to determination of the relative DNA content of Feulgen stained nuclei. The principles of the method are described and comparative measurements are presented. The determination is based on the measurement of areas at preset intervals of transmittance, taking into consideration the necessary correction of extinction values in relation to area and applying a correction for background. The data obtained can be used for the quantitative determination of DNA, and to provide analytical information for quantitation of structure.Comparison of the mean of extinctions as recorded with the scanning photometric method and those recorded with the television texture analysis method, with which 43 nuclei were investigated in transmittance steps of 0.01, revealed a coefficient of correlation of 0.9938.     

Determination of selenium in the human brain by graphite furnace atomic absorption spectrometry

For the investigation of neurological disorders, a development of simple and accessible methods for determining selenium in human brain samples is required. We devised a method of determining selenium using graphite furnace atomic absorption spectrometry (GFAAS). An electrodeless discharge lamp provided the sufficient sensitivity to determine brain selenium. The matrix interferences were avoided by using high temperature, a prolonged pyrolysis step, and a palladium matrix modifier. The technique of standard addition was used to evaluate the sample concentrations. The accuracy of the method was confirmed by a bovine liver reference material. The detection limit of selenium was 0.04 ng. The determined selenium concentrations of human brain cortex and white matter were higher than those of putamen (115–155 and 206–222 ng/g wet wt, respectively). These GFAAS values agreed with those obtained by fluorometric analysis (r=0.91,n=10). Moreover, the GFAAS values were compatible to those reported by other researchers (99–274 ng/g wet wt), in which selenium concentrations in putamen also tended to be higher than the other two regions. We conclude that GFAAS is useful for selenium analysis in brain samples.     

Measurement of endogenous lithium levels in serum and urine by electrothermal atomic absorption spectrometry: a method with potential clinical applications

A highly sensitive flameless atomic absorption method has been adapted for the determination of endogenous trace lithium levels in serum and urine. With ammonium nitrate as the only matrix modifier, serum levels of Li as low as 0.03 mumol/liter are measured accurately and there is no requirement for standard additions. The need for background correction during analysis was clearly established, and tungsten and Zeeman-effect background corrections were compared. The tungsten correction offered superior sensitivity and linearity of standards. Recoveries in urine and serum average 94.8 +/- 7.7 and 95.3 +/- 6.1% (+/- SD), respectively. The endogenous serum Li levels were 0.16 +/- 0.08 mumol/liter for normal subjects dwelling in the Denver metropolitan area. The mean 24-h excretion rate was 5.24 +/- 1.4 mumol/day. The mean fractional excretion of endogenous Li (clearance Li/clearance creatinine) was 23.2 +/- 3.0%, a value similar to values published for exogenously administered Li and measured by conventional methods.     

Reference values of selenium in plasma in population from Barcelona. Comparison with several pathologies

Plasma selenium reference values from healthy donors in the metropolitan area of Barcelona are determined. A random sample from 156 healthy adults (control group) is analysed by using electrothermal atomic absorption spectrometry with Zeeman effect background correction.

The relationship between several pathologies and Se content is also evaluated. Se content from 64 samples from subjects with chronic renal failure and 54 from subjects suffering from several malignancies are determined and the results are compared to the reference values. Moreover, Se contents are determined and compared in two groups of children, healthy (19 samples) and children of mothers infected with HIV-1 (16 samples).

In the control group, Se plasma concentration ranges between 50 and 145 μg · L⁻¹ (82.2 ± 17.5 μg · L⁻¹). Significantly lower values are found in the two pathologies studied (malignancy and chronic renal failure), compared to the control group. However, no significant differences in Se content are found between the two groups studied regarding malignancy and chronic renal failure.

In children of mothers infected with HIV-1, Se status is significantly lower than that of healthy children.     

Determination of norfloxacin in rat liver perfusate using capillary electrophoresis with laser-induced fluorescence detection

A capillary zone electrophoresis method has been developed for the direct determination of norfloxacin in the physiological perfusate of isolated rat liver. Norfloxacin and the internal standard triamterene were detected using laser-induced fluorescence (LIF) detection with the excitation and emission wavelength of 325 and 435 nm, respectively. The background electrolyte (BGE) was 50 mM phosphate buffer (pH 4.6). The effect of pH and concentration of BGE on the electrophoretic migration and fluorescence response of analytes were examined. Calibration curves were linear over a wide range of 0.01-100 microg/mL. The limit of quantitation was 0.01 microg/mL. The intra- and inter-day relative standard deviation was 3.7%, or less, and the accuracy was 93.2% of the nominal concentration. No endogenous substances were found to interfere. The method was used to characterize the steady-state and transient pharmacokinetics of norfloxacin in the rat liver.