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Determination of selenium in biological samples with an energy-dispersive X-ray fluorescence spectrometer
Author’s Accepted Manuscript Determination of selenium in biological samples with an energy-dispersive X-Ray fluorescence spectrometer Xiaoli Li, Zhaoshui Yu www.elsevier.com/locate/apradiso
PII: DOI: Reference:
S0969-8043(16)30030-6 http://dx.doi.org/10.1016/j.apradiso.2016.02.010 ARI7402
To appear in: Applied Radiation and Isotopes Received date: 4 November 2015 Revised date: 29 January 2016 Accepted date: 2 February 2016 Cite this article as: Xiaoli Li and Zhaoshui Yu, Determination of selenium in biological samples with an energy-dispersive X-Ray fluorescence spectrometer, Applied Radiation and Isotopes, http://dx.doi.org/10.1016/j.apradiso.2016.02.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of selenium in biological samples with an energy-dispersive X-Ray fluorescence spectrometer Xiaoli Lia, Zhaoshui Yub* a
Tianjin Institute of Geology and Mineral Resources,China;
b
Institute of Geophysics and Geochemical Exploration, Chinese Academy of Geological
Sciences,China * Corresponding author. email: [email protected] Abstract Selenium is both a nutrient and a toxin. Selenium—especially organic selenium—is a core component of human nutrition. Thus, it is very important to measure selenium in biological samples. The limited sensitivity of conventional XRF hampers its widespread use in biological samples. Here, we describe the use of high-energy(100Kv,600W) linearly polarized beam energy-dispersive
X-Ray
fluorescence
spectroscopy
(EDXRF)
in
tandem
with
a
three-dimensional optics design to determine 0.1-5.1 μg g−1 levels of selenium in biological samples. The effects of various experimental parameters such as applied voltage, acquisition time, secondary target and various filters were thoroughly investigated. The detection limit of selenium in biological samples via high-energy(100kV,600W) linearly polarized beam energy-dispersive X-ray fluorescence spectroscopy was decreased by one order of magnitude versus conventional XRF(Nicholas G et al.,2012) and found to be 0.1 μg/g. To the best of our knowledge, this is the first report to describe EDXRF measurements of Se in biological samples with important implications for the nutrition and analytical chemistry communities. Keywords: selenium, biological samples, high-energy polarized X-Ray fluorescence spectrometer, limit of detection.
Introduction The selenium concentration in biological samples is an important issue because it has a very narrow therapeutic window. That is, the concentration that is both safe and effective is narrow. At the proper concentration, Se is a nutrient, but at too high of a concentration it is toxic while low concentrations can lead to malnutrition( Bidari et al., 2007) Indeed, recommended allowances of Se
in foodstuffs fall in a very narrow range: consumption of food containing below 0.1 mg kg–1 will result in deficiency, whereas dietary levels above 1 mg kg–1 will lead to toxic events such as gastric distress or problems with hair and nails loss and poor productivity(Fordyce,2005). Since Schwarz and Foltz (1957) demonstrated the nutrition of selenium, more and more attention has
been given to it s unique physiological role. Many studies have indicated that selenium is a trace element essential to organisms with anti-cancer activity. It can scavenge free radicals and has anti-oxidation and anti-aging effects(Panigati M. 2007, Rotruck et al. 1969 ). It can protect cell membrane structures and function, and enhances immunity. Studies have shown that the physiological activity of selenium is closely related to the selenium form. Selenium—especially organic selenium—is a core component of human nutrition. While animal-based foods can supply Se in the food chain, plant sources are much more common. Therefore, biological selenium resources directly affect human selenium nutritional status. Thus, measuring selenium in biological samples including foodstuffs is very important to prevent both overdose and underdose effects. The abundance of Se in biological samples is only 10-8−10--6 and it is often difficult to measure such low concentrations especially in complex matrices such as food—this hampers most spectroscopic techniques. High sensitivity is required for the determination method of selenium, and various methods have been used to measure selenium in biological samples. On-line chemical vapor generation-ICP OES (Éder José dos Santos et al..,2009), hydride generation–inductively coupled plasma mass spectrometry(Moor et al..,2000), dynamic reaction cell inductively coupled plasma mass spectrometry (DRC-ICP-MS) after microwave digestion(Yi Hua et al.,2014) , electrothermal atomic absorption spectrometry (Anu and Anatoly B,2006), and hydride generation-atomic fluorescence
spectrometry(Qiu-Xiang,2010)
potentially
have
high
sensitivity
and
low interference. However, these methods also suffer from complicated sample preparation steps including dry ashing and wet ashing acid digestion process—this can lead to a loss of volatile elements. While neutron activation analysis has simple sample preparation (Marian,1988 ),but the analysis is time-consuming and expensive.
X-ray fluorescence (XRF) is a non-destructive measurement approach that offers simple sample analysis. However, conventional XRF has been hampered by poor elemental sensitivity due to high background from instrumental geometries and matrix effects. In XRF, the relatively
low sensitivity often makes a decomposition and preconcentration step mandatory. One tool to increase sensitivity is energy-dispersive XRF. This has been used for zinc, iron and selenium analyses in whole grain wheat with a Se limit of detection of 2 mg kg–1(Nicholas G et al.,2012). Previously EDXRF has been used for trace selenium measurements in organic and biological matrices after rapid acid digestion and thin-film deposition(Sotirois et al.,1980). C. Shenberg et al.(1988) determined selenium in human serum (0.75 μl samples) via EDXRF, and the minimum limit of detection is 0.06 μg g−1 . E. Margui et al.(2010) explored the possibilities of several analytical approaches combined with total reflection X-ray (TXRF) spectrometry for soil Se determinations. A dispersive liquid−liquid microextraction procedure (DLLME) was used before the TXRF analysis of the soil digest. The limit of detection (LOD) using this analytical methodology (0.05 mg/kg of Se) was comparable to or lower than those obtained in previous studies. Selenium has also been measured in biological sample by hydride evolution and wavelength dispersive X-ray fluorescence spectrometry; the limits of detection was 0.13 μg g-1 (Bohmer and Psotta ,1990). Suitable limits of detection have also been achieved for the determination of Se in biological and fluid samples with and without preconcentration strategies (Bellisola et al.,1999; Griessel et al.,2006; Mukhtar et al.,1991). Here, we used EDXRF with a high-energy polarized spectrometer to determine 0.1-5.1 μg g−1 levels of selenium in biological samples. This process uses minimal sample preparation steps and is compatible with a general EDXRF spectrometer. To the best of our knowledge, this is the first report to use EDXRF with a high-energy polarized spectrometer to measure Se in biological samples. The results have important implications for the dietary and analytical chemistry community. Materials and Methods We used an Epsilon 5 EDXRF (Holiland, Panalytical com.) with a maximum voltage of 100 kV, maximum power of 600 W, and 24 mA, ScW synthetic anode. This was configured with 15 secondary targets，PAN32 Ge-detector, a side window, and a beryllium window thickness of 150 μm. The activity area of the crystal is 30 mm2, and the resolution for MnKαis less than 140 eV. The efficient energy detection range is 0.7-100 keV; the detector is cooled with liquid nitrogen. The WinAxil(based on AXIL) software package provided with the instrument was used for evaluation of the EDXRF spectra. The data of peak fitting, integral peak area, net peak area and
background signal were obtained directly from the deconvolution software. And the background signal is subtracted from the integral peak area automatically. The instrument operation condition is given in table 1. Table 1 Spectrometer operation conditions. Target
Type
Zr
Secondary target
Mo B4C Al2O3 KBr W CaF2 Ti Ge KBr Zr
Secondary target Barkla target Barkla target Secondary target Secondary target Secondary target Secondary target Secondary target Secondary target Secondary target
Filter Al100, Al500, Cu100, Zr125, Mo250 none None none none none none none none none none
Counting time(s)
Analyte
1000
Se
500 500 500 500 500 500 500 500 500 500
Se Se Se Se Se P,K Ca Fe As Br
Sample preparation We used 30 certified biological materials including liver, huangqi, ginseng, spinach, milk powder, wheat flour, rice flour, corn flour, soybean powder, cabbage, tea, chicken, and apple. These were prepared by Institute of Geophysics and Geochemical Exploration(IGGE), China and were used as calibrators. To make a calibration curve, 5 synthetic standards were made with certified referenc materials. First, the biological sample was pulverized to 180μm (80 mesh), and dried at 60℃ 24hrs in the oven. Next, 6.0g of sample is weighed and subjected to 1500kN to form pellet using a liquid press developed in house.
Results and discussion Generally hundreds of mgs or several grams of biological samples were used to press pellet under 150-400kN pressure. The powder on the surface of this pellet(pressed under 150-400kN pressure) is easily detached. This will lead to contamination of the EDXRF measurement chamber and decrease th longevity and stability of the instrument. The pellet is not stable and is easily deformed. Thus, the time between sample preparation and determination should be as short as
possible to prevent deformation of the specimen surface (Queralt et al.,2005; Margui et al.,2005). The addition of a binder can make the pellet more stable, however it is critical that the source material be homogeneous but without excessive sample preparation. The approach we used here accomplished this. We used a high-pressure pressed powder pellet technique, without a binder, to solve the sample preparation issues related to biological samples XRF analysis. This is a major technological breakthrough for XRF sample preparation. This technique eliminates the need for a binder, thus increasing the analytical sensitivity, decreasing the relative standard deviations, and improving detection limits for most of components. Biological samples are mainly composed of C, N, H, O et al. This causes significant scatter of primary radiation from the tube. The high background of the biological specimen also arose from the high secondary scattering of the X-ray source by organic matrices. Therefore, the sensitivity of general X-Ray fluorescence spectrometry is insufficient to determine Se levels in biological samples. To solve this, we employed the three-dimensional geometrical optics system in the Epsilon 5 instrument—here, the optical pathway is not planar, but consists of two mutually perpendicular planes(see figure1). Background reduction by excitation with polarized X-ray radiation is based on the anisotropy of the atomic scattering cross section for polarized X-ray radiation.( Dazubay et al.,1974). Unpolarised X-rays emitted from an X-ray tube, on undergoing 90˚ scatter by a low-Z target, becomes highly plane polarized. This scattered polarized beam excites fluorescent X-rays in the sample but cannot rescatter at an angle of 90˚, so this becomes the optimum position for placing a detector to receive the minimum amount of tube line scatter. Thus, if the sample to be analysed is excited with linear polarized X-ray radiation, only the fluorescence radiation excited in the sample, and ideally none of the primary radiation scattered by the sample, reaches a suitably positioned detector(Stephens and Calder,2004 ). This novel design decreased background scattering by an order of magnitude, and the ratio of the peak to the background increased. This is critical for measuring trace elemental analysis. Indeed, this approach was used by Hiroyuki et al.,(2005) to successfully determine Cd in rice down to 20 ng/g .
1. Selection of secondary targets Another advantage of EDXRF is the concurrent analysis of 15 secondary targets. The 15 pure metal targets or compound targets were: Al2O3, B4C, W, CeO2, BaF2, CsI, Ag, Mo, Zr, KBr, Ge, Fe, Ti, CaF2, and Al. All of these were suitable for the excitation of specific elements or small groups of adjacent elements. The secondary targets can be classified into 2 types: Fluorescent target and Barkla target. The fluorescent targets are targets of which the fluorescence of the elements in the target is used to excite the sample. Barkla targets(Al2O3 and B4C ) are targets of which the scattered tube radiation is used to excite the sample. The Barkla target can be used for the excitation of elements with Z>26. This gave LODs over 0.1 μg/g. The use of a secondary target lowers the background, improves the peak-to-background ratio, and increases the LOD 5-10 times versus direct excitation. These are used to produce fluorescence lines that act as near-monochromatic secondary sources at slightly higher energies than the absorption edges of the analytical lines to be excited. This minimizes interference from the coexisting elements. For Se measurements in plants, SrF2 is the first choice for a secondary target. This is because the energy of the SrKα (14.14 keV) is only slightly higher than the absorption edges of the analytical lines of SeKα (12.65 keV). This offers the most efficient excitation for SeKα. However, because there is no SrF2 target available, we used Zr, Mo, W, KBr and the 2 Barkla target for Se excitation. Thus, six targets were used to scan the spectrum of the Se in GSB28(biological certified reference material of prawn) with selenium content of 5.1 μg g−1. The highest peak-to-background ratio occurs with W, B4C, Zr, KBr, Al2O3 and Mo. The Se spectrum is excited by different secondary targets. This results in three components of emitted X-rays from the general second targets: 1) the characteristic X-ray emitted from the secondary targets; 2) the scattering lines of the primary continuum of the tube, and 3) scattering lines from the X ray tube target characteristic X ray. For the W target, the WLγ1 from the secondary target and scattering line from the X-ray tube anode characteristic X ray are seriously imposed on the Se spectrum. When Barkla targets Al2O3 and B4C were used, the WLγ1 emitted from the tube are strongly scattered on Al2O3 and B4C —this seriously interferes with Se analysis. The energy of BrKα (11.907 keV) is slightly higher than the energy of the SeKα line (11.22 keV), but the Se was tailed with Br and the BrKα line interfered with the Se analysis(see figure2). So the use of Barkla target Al2O3 and B4C, secondary targets of W and KBr was discarded. The peak-to-background ratio of
Mo target are lower than Zr target. Thus, pure metallic Zr is used as the secondary target.
2. Spectrum acquisition time of selenium For the determination of trace selenium (10-8−10--6) in biological samples, prolonging the measurement time can reduce the statistical error, increase the precision and improve the limit of detection of the method. Thus, 0.6 μg/g Se sample was measured for 100, 200, 300, 400, 500, 600, 800 and 1000 seconds with five replicates each. The relative standard deviation (RSD) was calculated, and it decreased from 3.63% at 100s to0.71% at 1000s (Table 2). Thus, the spectrum acquisition time of selenium is set at 1000 sec because it offered the best tradeoff between RSD and sample throughput. Table 2. The spectrum acquisition time and relative standard deviation. Time (s)
RSD(%)
100
3.63
200 300 400 500 600 800 1000
2.20 2.17 1.25 1.19 1.17 0.81 0.71
3. The relation between the voltage and current applied and the intensity of selenium The voltage applied is usually 3-10 times that of the excitation potential of the analyte. At 600 W, a 0.6 μg/g Se sample was measured from 25 kV to 100 kV . The energy of K absorption edge of Se is 12.65 keV equals to the critical excitation potential of selenium. However, when the applied voltage is below the critical excitation potential of selenium, the selenium can not be excited, and the Se intensity is almost zero. As voltage is increased, the net peak area of the selenium increased substantially. When the applied voltage is maximum (about 9 times the excitation potential of SeKα), the net peak area of the selenium is maximized. The intensity of SeKα below 100 kV was about 37-fold that of SeKα under 25 kV. Under the optimal excitation voltage 100 kV , lowering the current of the X-ray tube from 6 mA to 1 mA, the intensity of net peak area is decreased from 2.24 cps mA -1 to 1.72 cps mA -1 and no dead time was set in the
experiment, see table3. So, the high voltage and current is beneficial for the determination of trace Se in biological samples.
Table 3. The relation between the current applied and the intensity of selenium Current(mA)
Net peak intensity (cps mA -1)
1
1.72
2
1.86
3
1.98
4
2.05
5
2.14
6
2.24
4. Selection of filters Filters used in EDXRF can not only efficiently decrease background and interference from the primary spectrum, but can also decrease dead time. Here, the Zr target is used as a secondary target. This is combined with no filter, an Al100 filter, an Al500 filter, a Cu100 filter, a Mo250 filter, or a Zr125 filter for the determination of biological sample GBS28 with a known Se concentration of 5.1 μg/g. The peak-to-background ratio of GSB28, the average intensity of five consecutive measurements as well as the RSD are shown in Table 4. Table 4 The measurement property Zr secondary target with different filters Measurement condition
P/B ratio
Average net peak intensity of 5 measurements (cps mA -1)
RSD of 5 measurements
Zr+ none Zr+ Al100 Zr+ Al500 Zr+ Cu100 Zr+ Mo250 Zr+ Zr125
3.10 2.98 3.01 3.04 2.786 3.24
10.097 9.594 7.747 3.152 0.5158 1.9488
0.057 0.091 0.072 0.030 1.80 5.25
The net peak area of selenium is highest without a filter. The peak-to-background ratio is highest when Zr125 is used, but the net peak area is lower, and the RSD is high. For these reason, Se measurements did not use a filter. The percentage of dead time is affected by two main factors: incoming count rates and pulse processing time( can not changed by user). As the total incoming
count rates of selenium is not high, so it was not mandatory to lowering the current or using filter to decrease dead time.
4. The correction of matrix effect The Compton scattering line of ZrKα was used as an internal standard to calibrate the absorption effect and the variation in the density of different biological samples. The line overlap of As and Br and the matrix interference from P, K, Ca, and Fe were corrected using empirical coefficients. However, the ideal Se results can not be achieved via these coefficients. For the determination of trace selenium in biological samples, we used a region of interest (ROI) method, which was simple and effective. The starting energy and the energy of termination are 11.107-11.39 kev, and the linear correlation coefficient of SeKαafter regression is 0.9935, see figure3. The limit of detection (LOD) is an important performance parameter to characterize XRF instruments. The limit of detection is calculated as 3 times of the standard deviation of measurements of 10 different pellets of the same samples with selenium concentration of 0.6 μg/g . The limit of detection is related to the matrix of the sample. The scattering of the background intensity and the sensitivity of the analytes can be changed because of the different components and content of the sample, and the detection limit is also different. The proposed methodology to evaluate LODs, on the basis of the standard deviation of repeated samples measurements, has the advantage of including most of source of measurement errors giving a more robust estimate. The LOD of Se in biological samples was 0.1 μg/g(measurement time of 1000s), one order of magnitude lower compared with that of conventional XRF(Nicholas G et al.,2012) in the measurement time of 105s 5. Verification of the method The developed method was verified by the measurements of unknown samples with microwave digestion ICP-MS method. The EDXRF measured values agree with values measured by ICP-MS, see table5. So the method is viable to the determination of
0.1-5.1 μg g−1 levels of
selenium in biological samples. Table 5 The comparison of EDXRF measurement value with values of ICP-MS (μg/g). Sample kelp suringar
EDXRF value 3.63±0.085 2.36±0.054
ICP-MS 3.37±0.044 2.13±0.025
agar laver oyster yam lentils oyster mushroom mushroom fuling flammulina velutipes
1.85±0.045 3.73±0.13 0.65±0.031 0.27±0.0089 0.12±0.0098 0.78±0.036 0.45±0.023 0.18±0.0089 0.26±0.0088
1.93±0.029 3.82±0.076 0.73±0.022 0.32±0.0064 0.14±0.0032 0.73±0.023 0.42±0.011 0.15±0.0045 0.23±0.0062
Conclusion We used a high-pressure pressed powder pellet technique with EDXRF to measure selenium in biological samples. This is a major technological breakthrough for XRF sample preparation. And the performance of the measurement is substantially improved. We used a three-dimensional geometrical optics system to reduce the high background arising from scattering of the primary spectrum of the X-ray tube and second scattering radiation from the specimen. Se concentrations from 0.1 to 5.1 μg/g can be accurately determined. The LOD of Se in biological samples was 0.1 μg/g(measurement time of 1000s), one order of magnitude lower compared with that of conventional XRF(Nicholas G et al.,2012) in the measurement time of 105s. To the best of our knowledge, this is the first report to use EDXRF to measure Se in food samples. This approach is rapid and very sensitive. Relative to competing technologies that require a completely aqueous or dissolved sample, EDXRF can use real world samples with only slight sample preparation. Furthermore, because the analysis time is relatively short, this approach could be used for online analysis of foodstuffs to prevent toxic overdoses or underdoses. Future work will utilize this system in real-world samples including fresh groceries. Acknowledgement We wish to thank for the sponsor of the National major scientific instruments and equipment development projects -Study on the application of WED-XRF in the analysis of geological prospecting samples (project number : 2012YQ05007606) on the experiments described in this paper. Reference Anu Viitak , Anatoly B. Volynsky, 2006. Simple procedure for the determination of Cd, Pb, As and Se in biological samples by electrothermal atomic absorption spectrometry using colloidal Pd modifier.Talanta
70 ,890–895. Bellisola, G.,Pasti, F., Valdes, M., Torboli, A, 1999. The use of total-reflection X-ray fluorescence to track the metabolism and excretion of selenium in humans . Spectrochim. Acta, Part B 54, 1481–1485. Bidari, A., Jahromi, E. Z., Assadi, Y., Hosseini, M. R. M. ,2007.Microchem. J. 87, 6–12. Bohmer R.G. and P.K.Psotta. 1990. Determination of arsenic, antimony and selenium in biological samples by hydride evolution and X-ray fluorescence Spectrometry, Fresenius’ journal of Analytical Chemistry,336,226-231 Fordyce, F.,2005. Selenium Deficiency and Toxicity in the Environment; British,Geological Survey; Elsevier: London, Chapter 15, pp 373-415. Éder José dos Santos , Amanda Beatriz Herrmann etc,2009. Determination of Se in biological samples by axial view inductively coupled plasma optical emission spectrometry after digestion with aqua regia and on-line chemical vapor generation.Spectrochimica Acta Part B 64, 549–553. Griessel, S., Mundry, R.,Kakuschke, A., Fonfara, S., Siebert, U., Prange,A., 2006. The use of total-reflection X-ray fluorescence to track the metabolism and excretion of selenium in humans.Spectrochim. Acta, Part B 61, 1158– 1165. Hiroyuki N., Ryoko, Akiko H. et al.,2005. Determination of Cd at sub-ppm level in brown rice by X-ray fluorescence analysis based on the Cd Kα line. Adv. X-ray .36,235-247. Marian Czauderna,1988.Appl. Determination of Selenium in Biological Materials by Neutron Activation Analysis. Applied Radiat &.Isot.47(8):735-737. Margui E., M.Hidalgo,I.Queralt, 2005. Multielemental fast analysis of vegetation samples by wavelength dispersive X-Ray fluorescence spectrometry: Possibilities and Drawbacks.Spectrochimica Acta PartB: Atomic pectroscopy.60,1363-1372. Margui E., G.H.Floor, M.Hidalgo et al.2010. Analytical Possibilities of Total Reflection X-ray Spectrometry (TXRF) for Trace Selenium Determination in Soils. Anal. Chem., 82, 7744–7751. Moor, J.W.H. Lam, R.E. Sturgeon, J.,2000. A novel introduction system for hydride generation–inductively coupled plasma mass spectrometry: determination of selenium in biological materials.Anal. At. Spectrom. 15 , 143–149. Mukhtar, S., Haswell, S. J., Ellis, A.,Hawke, D. T., 1991.
Application of Total-reflection X-ray fluorescence
spectrometry to elemental determination in water, soil and sewage sludge. Analyst , 116, 333–338. Nicholas G. Paltridge, Paul J. Micham, J. Ivan Ortiz-Monasterio et al., 2012. Energy-dispersive X-ray fluorescence spectrometry as a tool for zinc, iron and selenium analysis in whole grain wheat. Plant and soil . 361, 261-269. Panigati M. Falciola L. Mussini P. 2007. Determination of selenium in Italian rices by differential pulse cathodic stripping voltammetry. Food Chemistry,105,1091-1098. Qiu-Xiang Zhao, Yu-Wei Chen, Nelson Belzile, Mohui Wang, 2010. Low volume microwave digestion and direct determination of selenium in biological samples by hydride generation-atomic fluorescence spectrometry. Analytica Chimica Acta 665 , 123–128. Queralt I.,M.Ovejero,M.L.Carvalho,A.F.Marques,J.M.Labres, 2005. Quantitative determination of essential and trace element content of medicinal plants and their infusions by XRF and ICP techniquesX-Ray Spectrom.34,213-217. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG,Hoekstra WG (1969) Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588–590 Schwarz K, Foltz CM .1957. Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J Am Chem Soc.79:3292–3293
Shamberger RJ, Frost DV
Shenberg C., M.Mental, T. Izak-Biran et al. 1988. Rapid and simple determination of Selenium and other trace elements in very small blood samples by XRF. Biological trace elements research, 16,87-91. Sotirois E.Raptis, Wolfhard Wegscheider, Guenter knapp et al., 1980. X-ray fluorescence determination of trace selenium in organic and biological matrices. Anal. Chem., 52, 1292–1296. T.G. Dazubay, B.V. Jarret, J.M. Jaklevic,1974.Background reduction in X-ray fluorescenc spectra using polarization. Nucl. Instrum. Meth.115, 297-299. W.E. Stephens, A. Calder. 2004. Analysis of non-organic elements in plant foliage using polarized X-ray fluorescence spectrometry.Anal. Chim.Acta 527,89-96 Yi Hua Wei, Jin Yan Zhang, Da Wen Zhang, Lin Guang Luo, Tian Hua Tu., 2014. Simultaneous determination of Se, trace elements and major elements in Se-rich rice by dynamic reaction cell inductively coupled plasma mass spectrometry (DRC-ICP-MS) after microwave digestion.Food Chemistry 159, 507–511.
Figure 1. The scheme of the three-dimensional geometrical optics system in the Epsilon 5
Figure2 The SeKαspectrum excited by Al2O3 Barkla target and its interferences
Figure 3
Selenium measured value versus certified value
Highlights 1. The
high-energy(100Kv,600W)
linearly
polarized
beam
energy-dispersive X-Ray fluorescence spectroscopy (EDXRF) in tandem with a three-dimensional optics design was used to determine 0.1-5.1 μg g−1 levels of selenium in biological samples.
2. High-pressure pressed powder pellet technique(1500 kN) is used for sample preparation and the pellet is more stable, efficiently avoid the contamination of the measuring chamber.
3. The detection limit of selenium in biological samples via high-energy linearly polarized beam energy-dispersive X-ray fluorescence spectroscopy was decreased by one order of magnitude versus conventional XRF and found to be 0.1 μg/g.
PII: DOI: Reference:
S0969-8043(16)30030-6 http://dx.doi.org/10.1016/j.apradiso.2016.02.010 ARI7402
To appear in: Applied Radiation and Isotopes Received date: 4 November 2015 Revised date: 29 January 2016 Accepted date: 2 February 2016 Cite this article as: Xiaoli Li and Zhaoshui Yu, Determination of selenium in biological samples with an energy-dispersive X-Ray fluorescence spectrometer, Applied Radiation and Isotopes, http://dx.doi.org/10.1016/j.apradiso.2016.02.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of selenium in biological samples with an energy-dispersive X-Ray fluorescence spectrometer Xiaoli Lia, Zhaoshui Yub* a
Tianjin Institute of Geology and Mineral Resources,China;
b
Institute of Geophysics and Geochemical Exploration, Chinese Academy of Geological
Sciences,China * Corresponding author. email: [email protected] Abstract Selenium is both a nutrient and a toxin. Selenium—especially organic selenium—is a core component of human nutrition. Thus, it is very important to measure selenium in biological samples. The limited sensitivity of conventional XRF hampers its widespread use in biological samples. Here, we describe the use of high-energy(100Kv,600W) linearly polarized beam energy-dispersive
X-Ray
fluorescence
spectroscopy
(EDXRF)
in
tandem
with
a
three-dimensional optics design to determine 0.1-5.1 μg g−1 levels of selenium in biological samples. The effects of various experimental parameters such as applied voltage, acquisition time, secondary target and various filters were thoroughly investigated. The detection limit of selenium in biological samples via high-energy(100kV,600W) linearly polarized beam energy-dispersive X-ray fluorescence spectroscopy was decreased by one order of magnitude versus conventional XRF(Nicholas G et al.,2012) and found to be 0.1 μg/g. To the best of our knowledge, this is the first report to describe EDXRF measurements of Se in biological samples with important implications for the nutrition and analytical chemistry communities. Keywords: selenium, biological samples, high-energy polarized X-Ray fluorescence spectrometer, limit of detection.
Introduction The selenium concentration in biological samples is an important issue because it has a very narrow therapeutic window. That is, the concentration that is both safe and effective is narrow. At the proper concentration, Se is a nutrient, but at too high of a concentration it is toxic while low concentrations can lead to malnutrition( Bidari et al., 2007) Indeed, recommended allowances of Se
in foodstuffs fall in a very narrow range: consumption of food containing below 0.1 mg kg–1 will result in deficiency, whereas dietary levels above 1 mg kg–1 will lead to toxic events such as gastric distress or problems with hair and nails loss and poor productivity(Fordyce,2005). Since Schwarz and Foltz (1957) demonstrated the nutrition of selenium, more and more attention has
been given to it s unique physiological role. Many studies have indicated that selenium is a trace element essential to organisms with anti-cancer activity. It can scavenge free radicals and has anti-oxidation and anti-aging effects(Panigati M. 2007, Rotruck et al. 1969 ). It can protect cell membrane structures and function, and enhances immunity. Studies have shown that the physiological activity of selenium is closely related to the selenium form. Selenium—especially organic selenium—is a core component of human nutrition. While animal-based foods can supply Se in the food chain, plant sources are much more common. Therefore, biological selenium resources directly affect human selenium nutritional status. Thus, measuring selenium in biological samples including foodstuffs is very important to prevent both overdose and underdose effects. The abundance of Se in biological samples is only 10-8−10--6 and it is often difficult to measure such low concentrations especially in complex matrices such as food—this hampers most spectroscopic techniques. High sensitivity is required for the determination method of selenium, and various methods have been used to measure selenium in biological samples. On-line chemical vapor generation-ICP OES (Éder José dos Santos et al..,2009), hydride generation–inductively coupled plasma mass spectrometry(Moor et al..,2000), dynamic reaction cell inductively coupled plasma mass spectrometry (DRC-ICP-MS) after microwave digestion(Yi Hua et al.,2014) , electrothermal atomic absorption spectrometry (Anu and Anatoly B,2006), and hydride generation-atomic fluorescence
spectrometry(Qiu-Xiang,2010)
potentially
have
high
sensitivity
and
low interference. However, these methods also suffer from complicated sample preparation steps including dry ashing and wet ashing acid digestion process—this can lead to a loss of volatile elements. While neutron activation analysis has simple sample preparation (Marian,1988 ),but the analysis is time-consuming and expensive.
X-ray fluorescence (XRF) is a non-destructive measurement approach that offers simple sample analysis. However, conventional XRF has been hampered by poor elemental sensitivity due to high background from instrumental geometries and matrix effects. In XRF, the relatively
low sensitivity often makes a decomposition and preconcentration step mandatory. One tool to increase sensitivity is energy-dispersive XRF. This has been used for zinc, iron and selenium analyses in whole grain wheat with a Se limit of detection of 2 mg kg–1(Nicholas G et al.,2012). Previously EDXRF has been used for trace selenium measurements in organic and biological matrices after rapid acid digestion and thin-film deposition(Sotirois et al.,1980). C. Shenberg et al.(1988) determined selenium in human serum (0.75 μl samples) via EDXRF, and the minimum limit of detection is 0.06 μg g−1 . E. Margui et al.(2010) explored the possibilities of several analytical approaches combined with total reflection X-ray (TXRF) spectrometry for soil Se determinations. A dispersive liquid−liquid microextraction procedure (DLLME) was used before the TXRF analysis of the soil digest. The limit of detection (LOD) using this analytical methodology (0.05 mg/kg of Se) was comparable to or lower than those obtained in previous studies. Selenium has also been measured in biological sample by hydride evolution and wavelength dispersive X-ray fluorescence spectrometry; the limits of detection was 0.13 μg g-1 (Bohmer and Psotta ,1990). Suitable limits of detection have also been achieved for the determination of Se in biological and fluid samples with and without preconcentration strategies (Bellisola et al.,1999; Griessel et al.,2006; Mukhtar et al.,1991). Here, we used EDXRF with a high-energy polarized spectrometer to determine 0.1-5.1 μg g−1 levels of selenium in biological samples. This process uses minimal sample preparation steps and is compatible with a general EDXRF spectrometer. To the best of our knowledge, this is the first report to use EDXRF with a high-energy polarized spectrometer to measure Se in biological samples. The results have important implications for the dietary and analytical chemistry community. Materials and Methods We used an Epsilon 5 EDXRF (Holiland, Panalytical com.) with a maximum voltage of 100 kV, maximum power of 600 W, and 24 mA, ScW synthetic anode. This was configured with 15 secondary targets，PAN32 Ge-detector, a side window, and a beryllium window thickness of 150 μm. The activity area of the crystal is 30 mm2, and the resolution for MnKαis less than 140 eV. The efficient energy detection range is 0.7-100 keV; the detector is cooled with liquid nitrogen. The WinAxil(based on AXIL) software package provided with the instrument was used for evaluation of the EDXRF spectra. The data of peak fitting, integral peak area, net peak area and
background signal were obtained directly from the deconvolution software. And the background signal is subtracted from the integral peak area automatically. The instrument operation condition is given in table 1. Table 1 Spectrometer operation conditions. Target
Type
Zr
Secondary target
Mo B4C Al2O3 KBr W CaF2 Ti Ge KBr Zr
Secondary target Barkla target Barkla target Secondary target Secondary target Secondary target Secondary target Secondary target Secondary target Secondary target
Filter Al100, Al500, Cu100, Zr125, Mo250 none None none none none none none none none none
Counting time(s)
Analyte
1000
Se
500 500 500 500 500 500 500 500 500 500
Se Se Se Se Se P,K Ca Fe As Br
Sample preparation We used 30 certified biological materials including liver, huangqi, ginseng, spinach, milk powder, wheat flour, rice flour, corn flour, soybean powder, cabbage, tea, chicken, and apple. These were prepared by Institute of Geophysics and Geochemical Exploration(IGGE), China and were used as calibrators. To make a calibration curve, 5 synthetic standards were made with certified referenc materials. First, the biological sample was pulverized to 180μm (80 mesh), and dried at 60℃ 24hrs in the oven. Next, 6.0g of sample is weighed and subjected to 1500kN to form pellet using a liquid press developed in house.
Results and discussion Generally hundreds of mgs or several grams of biological samples were used to press pellet under 150-400kN pressure. The powder on the surface of this pellet(pressed under 150-400kN pressure) is easily detached. This will lead to contamination of the EDXRF measurement chamber and decrease th longevity and stability of the instrument. The pellet is not stable and is easily deformed. Thus, the time between sample preparation and determination should be as short as
possible to prevent deformation of the specimen surface (Queralt et al.,2005; Margui et al.,2005). The addition of a binder can make the pellet more stable, however it is critical that the source material be homogeneous but without excessive sample preparation. The approach we used here accomplished this. We used a high-pressure pressed powder pellet technique, without a binder, to solve the sample preparation issues related to biological samples XRF analysis. This is a major technological breakthrough for XRF sample preparation. This technique eliminates the need for a binder, thus increasing the analytical sensitivity, decreasing the relative standard deviations, and improving detection limits for most of components. Biological samples are mainly composed of C, N, H, O et al. This causes significant scatter of primary radiation from the tube. The high background of the biological specimen also arose from the high secondary scattering of the X-ray source by organic matrices. Therefore, the sensitivity of general X-Ray fluorescence spectrometry is insufficient to determine Se levels in biological samples. To solve this, we employed the three-dimensional geometrical optics system in the Epsilon 5 instrument—here, the optical pathway is not planar, but consists of two mutually perpendicular planes(see figure1). Background reduction by excitation with polarized X-ray radiation is based on the anisotropy of the atomic scattering cross section for polarized X-ray radiation.( Dazubay et al.,1974). Unpolarised X-rays emitted from an X-ray tube, on undergoing 90˚ scatter by a low-Z target, becomes highly plane polarized. This scattered polarized beam excites fluorescent X-rays in the sample but cannot rescatter at an angle of 90˚, so this becomes the optimum position for placing a detector to receive the minimum amount of tube line scatter. Thus, if the sample to be analysed is excited with linear polarized X-ray radiation, only the fluorescence radiation excited in the sample, and ideally none of the primary radiation scattered by the sample, reaches a suitably positioned detector(Stephens and Calder,2004 ). This novel design decreased background scattering by an order of magnitude, and the ratio of the peak to the background increased. This is critical for measuring trace elemental analysis. Indeed, this approach was used by Hiroyuki et al.,(2005) to successfully determine Cd in rice down to 20 ng/g .
1. Selection of secondary targets Another advantage of EDXRF is the concurrent analysis of 15 secondary targets. The 15 pure metal targets or compound targets were: Al2O3, B4C, W, CeO2, BaF2, CsI, Ag, Mo, Zr, KBr, Ge, Fe, Ti, CaF2, and Al. All of these were suitable for the excitation of specific elements or small groups of adjacent elements. The secondary targets can be classified into 2 types: Fluorescent target and Barkla target. The fluorescent targets are targets of which the fluorescence of the elements in the target is used to excite the sample. Barkla targets(Al2O3 and B4C ) are targets of which the scattered tube radiation is used to excite the sample. The Barkla target can be used for the excitation of elements with Z>26. This gave LODs over 0.1 μg/g. The use of a secondary target lowers the background, improves the peak-to-background ratio, and increases the LOD 5-10 times versus direct excitation. These are used to produce fluorescence lines that act as near-monochromatic secondary sources at slightly higher energies than the absorption edges of the analytical lines to be excited. This minimizes interference from the coexisting elements. For Se measurements in plants, SrF2 is the first choice for a secondary target. This is because the energy of the SrKα (14.14 keV) is only slightly higher than the absorption edges of the analytical lines of SeKα (12.65 keV). This offers the most efficient excitation for SeKα. However, because there is no SrF2 target available, we used Zr, Mo, W, KBr and the 2 Barkla target for Se excitation. Thus, six targets were used to scan the spectrum of the Se in GSB28(biological certified reference material of prawn) with selenium content of 5.1 μg g−1. The highest peak-to-background ratio occurs with W, B4C, Zr, KBr, Al2O3 and Mo. The Se spectrum is excited by different secondary targets. This results in three components of emitted X-rays from the general second targets: 1) the characteristic X-ray emitted from the secondary targets; 2) the scattering lines of the primary continuum of the tube, and 3) scattering lines from the X ray tube target characteristic X ray. For the W target, the WLγ1 from the secondary target and scattering line from the X-ray tube anode characteristic X ray are seriously imposed on the Se spectrum. When Barkla targets Al2O3 and B4C were used, the WLγ1 emitted from the tube are strongly scattered on Al2O3 and B4C —this seriously interferes with Se analysis. The energy of BrKα (11.907 keV) is slightly higher than the energy of the SeKα line (11.22 keV), but the Se was tailed with Br and the BrKα line interfered with the Se analysis(see figure2). So the use of Barkla target Al2O3 and B4C, secondary targets of W and KBr was discarded. The peak-to-background ratio of
Mo target are lower than Zr target. Thus, pure metallic Zr is used as the secondary target.
2. Spectrum acquisition time of selenium For the determination of trace selenium (10-8−10--6) in biological samples, prolonging the measurement time can reduce the statistical error, increase the precision and improve the limit of detection of the method. Thus, 0.6 μg/g Se sample was measured for 100, 200, 300, 400, 500, 600, 800 and 1000 seconds with five replicates each. The relative standard deviation (RSD) was calculated, and it decreased from 3.63% at 100s to0.71% at 1000s (Table 2). Thus, the spectrum acquisition time of selenium is set at 1000 sec because it offered the best tradeoff between RSD and sample throughput. Table 2. The spectrum acquisition time and relative standard deviation. Time (s)
RSD(%)
100
3.63
200 300 400 500 600 800 1000
2.20 2.17 1.25 1.19 1.17 0.81 0.71
3. The relation between the voltage and current applied and the intensity of selenium The voltage applied is usually 3-10 times that of the excitation potential of the analyte. At 600 W, a 0.6 μg/g Se sample was measured from 25 kV to 100 kV . The energy of K absorption edge of Se is 12.65 keV equals to the critical excitation potential of selenium. However, when the applied voltage is below the critical excitation potential of selenium, the selenium can not be excited, and the Se intensity is almost zero. As voltage is increased, the net peak area of the selenium increased substantially. When the applied voltage is maximum (about 9 times the excitation potential of SeKα), the net peak area of the selenium is maximized. The intensity of SeKα below 100 kV was about 37-fold that of SeKα under 25 kV. Under the optimal excitation voltage 100 kV , lowering the current of the X-ray tube from 6 mA to 1 mA, the intensity of net peak area is decreased from 2.24 cps mA -1 to 1.72 cps mA -1 and no dead time was set in the
experiment, see table3. So, the high voltage and current is beneficial for the determination of trace Se in biological samples.
Table 3. The relation between the current applied and the intensity of selenium Current(mA)
Net peak intensity (cps mA -1)
1
1.72
2
1.86
3
1.98
4
2.05
5
2.14
6
2.24
4. Selection of filters Filters used in EDXRF can not only efficiently decrease background and interference from the primary spectrum, but can also decrease dead time. Here, the Zr target is used as a secondary target. This is combined with no filter, an Al100 filter, an Al500 filter, a Cu100 filter, a Mo250 filter, or a Zr125 filter for the determination of biological sample GBS28 with a known Se concentration of 5.1 μg/g. The peak-to-background ratio of GSB28, the average intensity of five consecutive measurements as well as the RSD are shown in Table 4. Table 4 The measurement property Zr secondary target with different filters Measurement condition
P/B ratio
Average net peak intensity of 5 measurements (cps mA -1)
RSD of 5 measurements
Zr+ none Zr+ Al100 Zr+ Al500 Zr+ Cu100 Zr+ Mo250 Zr+ Zr125
3.10 2.98 3.01 3.04 2.786 3.24
10.097 9.594 7.747 3.152 0.5158 1.9488
0.057 0.091 0.072 0.030 1.80 5.25
The net peak area of selenium is highest without a filter. The peak-to-background ratio is highest when Zr125 is used, but the net peak area is lower, and the RSD is high. For these reason, Se measurements did not use a filter. The percentage of dead time is affected by two main factors: incoming count rates and pulse processing time( can not changed by user). As the total incoming
count rates of selenium is not high, so it was not mandatory to lowering the current or using filter to decrease dead time.
4. The correction of matrix effect The Compton scattering line of ZrKα was used as an internal standard to calibrate the absorption effect and the variation in the density of different biological samples. The line overlap of As and Br and the matrix interference from P, K, Ca, and Fe were corrected using empirical coefficients. However, the ideal Se results can not be achieved via these coefficients. For the determination of trace selenium in biological samples, we used a region of interest (ROI) method, which was simple and effective. The starting energy and the energy of termination are 11.107-11.39 kev, and the linear correlation coefficient of SeKαafter regression is 0.9935, see figure3. The limit of detection (LOD) is an important performance parameter to characterize XRF instruments. The limit of detection is calculated as 3 times of the standard deviation of measurements of 10 different pellets of the same samples with selenium concentration of 0.6 μg/g . The limit of detection is related to the matrix of the sample. The scattering of the background intensity and the sensitivity of the analytes can be changed because of the different components and content of the sample, and the detection limit is also different. The proposed methodology to evaluate LODs, on the basis of the standard deviation of repeated samples measurements, has the advantage of including most of source of measurement errors giving a more robust estimate. The LOD of Se in biological samples was 0.1 μg/g(measurement time of 1000s), one order of magnitude lower compared with that of conventional XRF(Nicholas G et al.,2012) in the measurement time of 105s 5. Verification of the method The developed method was verified by the measurements of unknown samples with microwave digestion ICP-MS method. The EDXRF measured values agree with values measured by ICP-MS, see table5. So the method is viable to the determination of
0.1-5.1 μg g−1 levels of
selenium in biological samples. Table 5 The comparison of EDXRF measurement value with values of ICP-MS (μg/g). Sample kelp suringar
EDXRF value 3.63±0.085 2.36±0.054
ICP-MS 3.37±0.044 2.13±0.025
agar laver oyster yam lentils oyster mushroom mushroom fuling flammulina velutipes
1.85±0.045 3.73±0.13 0.65±0.031 0.27±0.0089 0.12±0.0098 0.78±0.036 0.45±0.023 0.18±0.0089 0.26±0.0088
1.93±0.029 3.82±0.076 0.73±0.022 0.32±0.0064 0.14±0.0032 0.73±0.023 0.42±0.011 0.15±0.0045 0.23±0.0062
Conclusion We used a high-pressure pressed powder pellet technique with EDXRF to measure selenium in biological samples. This is a major technological breakthrough for XRF sample preparation. And the performance of the measurement is substantially improved. We used a three-dimensional geometrical optics system to reduce the high background arising from scattering of the primary spectrum of the X-ray tube and second scattering radiation from the specimen. Se concentrations from 0.1 to 5.1 μg/g can be accurately determined. The LOD of Se in biological samples was 0.1 μg/g(measurement time of 1000s), one order of magnitude lower compared with that of conventional XRF(Nicholas G et al.,2012) in the measurement time of 105s. To the best of our knowledge, this is the first report to use EDXRF to measure Se in food samples. This approach is rapid and very sensitive. Relative to competing technologies that require a completely aqueous or dissolved sample, EDXRF can use real world samples with only slight sample preparation. Furthermore, because the analysis time is relatively short, this approach could be used for online analysis of foodstuffs to prevent toxic overdoses or underdoses. Future work will utilize this system in real-world samples including fresh groceries. Acknowledgement We wish to thank for the sponsor of the National major scientific instruments and equipment development projects -Study on the application of WED-XRF in the analysis of geological prospecting samples (project number : 2012YQ05007606) on the experiments described in this paper. Reference Anu Viitak , Anatoly B. Volynsky, 2006. Simple procedure for the determination of Cd, Pb, As and Se in biological samples by electrothermal atomic absorption spectrometry using colloidal Pd modifier.Talanta
70 ,890–895. Bellisola, G.,Pasti, F., Valdes, M., Torboli, A, 1999. The use of total-reflection X-ray fluorescence to track the metabolism and excretion of selenium in humans . Spectrochim. Acta, Part B 54, 1481–1485. Bidari, A., Jahromi, E. Z., Assadi, Y., Hosseini, M. R. M. ,2007.Microchem. J. 87, 6–12. Bohmer R.G. and P.K.Psotta. 1990. Determination of arsenic, antimony and selenium in biological samples by hydride evolution and X-ray fluorescence Spectrometry, Fresenius’ journal of Analytical Chemistry,336,226-231 Fordyce, F.,2005. Selenium Deficiency and Toxicity in the Environment; British,Geological Survey; Elsevier: London, Chapter 15, pp 373-415. Éder José dos Santos , Amanda Beatriz Herrmann etc,2009. Determination of Se in biological samples by axial view inductively coupled plasma optical emission spectrometry after digestion with aqua regia and on-line chemical vapor generation.Spectrochimica Acta Part B 64, 549–553. Griessel, S., Mundry, R.,Kakuschke, A., Fonfara, S., Siebert, U., Prange,A., 2006. The use of total-reflection X-ray fluorescence to track the metabolism and excretion of selenium in humans.Spectrochim. Acta, Part B 61, 1158– 1165. Hiroyuki N., Ryoko, Akiko H. et al.,2005. Determination of Cd at sub-ppm level in brown rice by X-ray fluorescence analysis based on the Cd Kα line. Adv. X-ray .36,235-247. Marian Czauderna,1988.Appl. Determination of Selenium in Biological Materials by Neutron Activation Analysis. Applied Radiat &.Isot.47(8):735-737. Margui E., M.Hidalgo,I.Queralt, 2005. Multielemental fast analysis of vegetation samples by wavelength dispersive X-Ray fluorescence spectrometry: Possibilities and Drawbacks.Spectrochimica Acta PartB: Atomic pectroscopy.60,1363-1372. Margui E., G.H.Floor, M.Hidalgo et al.2010. Analytical Possibilities of Total Reflection X-ray Spectrometry (TXRF) for Trace Selenium Determination in Soils. Anal. Chem., 82, 7744–7751. Moor, J.W.H. Lam, R.E. Sturgeon, J.,2000. A novel introduction system for hydride generation–inductively coupled plasma mass spectrometry: determination of selenium in biological materials.Anal. At. Spectrom. 15 , 143–149. Mukhtar, S., Haswell, S. J., Ellis, A.,Hawke, D. T., 1991.
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Figure 1. The scheme of the three-dimensional geometrical optics system in the Epsilon 5
Figure2 The SeKαspectrum excited by Al2O3 Barkla target and its interferences
Figure 3
Selenium measured value versus certified value
Highlights 1. The
high-energy(100Kv,600W)
linearly
polarized
beam
energy-dispersive X-Ray fluorescence spectroscopy (EDXRF) in tandem with a three-dimensional optics design was used to determine 0.1-5.1 μg g−1 levels of selenium in biological samples.
2. High-pressure pressed powder pellet technique(1500 kN) is used for sample preparation and the pellet is more stable, efficiently avoid the contamination of the measuring chamber.
3. The detection limit of selenium in biological samples via high-energy linearly polarized beam energy-dispersive X-ray fluorescence spectroscopy was decreased by one order of magnitude versus conventional XRF and found to be 0.1 μg/g.