Analysis of nutrition-relevant trace elements in human blood and serum by means of total reflection X-ray fluorescence (TXRF) spectroscopy

doi:10.1016/j.sab.2009.03.019

Spectrochimica Acta Part B: Atomic Spectroscopy

Volume 64, Issue 4, April 2009, Pages 354-356

https://doi.org/10.1016/j.sab.2009.03.019 Get rights and content

Abstract

In clinical service laboratories, one of the most common analytical tasks with regard to inorganic traces is the determination of the nutrition-relevant elements Fe, Cu, Zn, and Se.

Because of the high numbers of samples and the commercial character of these analyses, a time-consuming sample preparation must be avoided.

In this presentation, the results of total reflection X-ray fluorescence measurements with a low-power system and different sample preparation procedures are compared with those derived from analysis with common methods like Atomic Absorption Spectroscopy (AAS) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS).

The results of these investigations indicate that the optimal total reflection X-ray fluorescence analysis of the nutrition-relevant elements Fe, Cu, Zn, and Se can be performed by preparing whole blood and serum samples after dilution with ultrapure water and transferring 10 μl of internally standardized sample to an unsiliconized quartz glass sample carrier with subsequent drying in a laboratory oven. Suitable measurement time was found to be 600 s.

The enhanced sample preparation by means of microwave or open digestion, in parts combined with cold plasma ashing, led to an improvement of detection limits by a factor of 2 for serum samples while for whole blood samples an improvement was only observed for samples prepared by means of microwave digestion. As the matrix elements P, S, Cl, and for whole blood Fe have a major influence on the detection limits, most probably a further enhancement of analytical quality requires the removal of the organic matrix.

However, for the routine analysis of the nutrition-relevant elements, the dilution preparation was found to be sufficient.

Introduction

Since the publication by Prange et al. [1], total reflection X-ray fluorescence has been established as a versatile tool for the analysis of trace elements in blood and serum samples. With this, the main application fields are the analysis of toxic elements [2] and medical investigations [3], [4]. Additionally, the improvement of detection limits and accuracies by means of enhanced sample preparation techniques, as published by Savage and Haswell [5], was a topic of several analytical studies.

However, one of the major analytical tasks in routine whole blood and serum analysis is the determination of nutrition-relevant trace elements. Among these, elements of crucial importance are Cu, Fe, Zn, and Se [6], [7], [8], [9]. The most established methods for the determination of these elements are Atomic Absorption Spectroscopy (ASS) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). As both methods have the disadvantages of a comparably complicated sample preparation and rather expensive analysis, total reflection X-ray fluorescence can be considered as a real analytical alternative.

The target of this study was to develop a method for the analysis of the nutrition-relevant elements with a minimum of sample preparation and measurement time, which still guarantees an accurate and reproducible analysis. For comparison purposes, additional measurements were performed with several certified reference standards, applying different preparation techniques, including microwave digestion and cold plasma ashing.

Section snippets

Instrumentation

All measurements presented in this paper were performed with the benchtop TXRF spectrometer “S2 PICOFOX” (Bruker AXS Microanalysis, Berlin, Germany). The technical specification of this instrument is summarized in Table 1.

Samples

For this study, the six differently certified reference standards “Serum ClinChek L1”, “Serum ClinChek L2”, “Serum ClinCal Calibrator”, “Whole blood ClinCal Calibrator” (Recipe) “Whole blood Seronorm L1” and “Whole blood Seronorm L2” (Sero) were analyzed. These reference

Accuracy

The results of the serum measurements are summarized in Table 2. Taking the confidence level of the reference values into account, it can be stated that the correspondence of the TXRF and the reference values are excellent for the elements Cu, Zn, and Se. For Fe a systematic overestimation, most probably caused by contamination, was observed.

A comparison of the reference standard confidence level values with the standard deviations of the TXRF measurements, estimated by means of a fivefold

Conclusions

Several whole blood and serum reference standards were analyzed by means of TXRF spectroscopy after simple dilution and enhanced digestions preparation. It can be concluded that for a routine analysis of the nutrition-relevant elements Fe, Cu, Zn, and Se the fast and simple dilution of the samples with ultrapure water is sufficient. 10 μl of the internal standardized samples should be prepared on unsiliconized quartz glass sample carriers. With this, measurement times of 600 s while applying a

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Trace elements exhibit essential functions in many physiological processes. Thus, for research focusing on trace element homeostasis and metabolism analytical methods allowing for multi-element analyses are fundamental. Small sample amounts may be a big challenge in trace element analyses especially if also other end points want to be addressed in the same sample. Therefore, the aim of the present study was to examine trace elements (iron, copper, zinc, and selenium) in murine liver tissue prepared by a RIPA buffer-based lyses method.
After centrifugation, lysates and pellets were obtained and trace elements were analyzed with TXRF in liver lysates. The results were compared to that obtained by a standard microwave-assisted acidic digestion with subsequent ICP-MS/MS analysis of the same liver tissue, liver lysates, and remaining pellets. In addition, trace element concentrations, determined in murine serum with both methods, were compared. For serum samples, both TXRF and ICP-MS/MS provide similar and highly correlating results. Furthermore, in liver lysate samples prepared with RIPA buffer, comparable trace element concentrations were measured by TXRF as with the standard digestion technique and ICP-MS/MS. Only marginal amounts of trace elements were detected in the pellets.
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Total X-ray Reflection Fluorescence (TXRF) has established itself as an easy-to-use analytical technique that offers quantification of trace elements from various sample types ranging from liquids to powdered solids [16,17]. Simultaneous multielemental TXRF quantification with the use of an internal standard has allowed for biomonitoring of blood trace elements to study the influence of environmental pollutants [18,19], nutrition [20,21] and elemental imbalances in various disorders [22–26]. Given the rich matrix of blood and its influence on quantification, several studies have also investigated effect of sample pre-treatment on quantification of whole blood, plasma and serum [20,21,27–32].
The interactions of gold nanoparticles (AuNPs) with blood represents the first systemic clearance barrier that these particles face in-vivo. Assessment of this interaction is of particular interest in evaluating the efficacy and kinetics of AuNP delivery in various tissues or organs. Total reflection X-ray fluorescence (TXRF) spectroscopy offers multielemental quantification of trace-level concentrations through internal standardization. In this work, we investigate different sample preparation procedures to determine an appropriate method that would allow accurate quantification of AuNPs in blood matrix. Whole blood, plasma and red blood cells samples were doped with La internal standard and certified Au solution. The samples were prepared as is and diluted with water in ratios of 1:1, 1:5 and 1:10. In order to extend the investigation to AuNPs, samples were also doped with 10 nm reference material AuNPs and diluted with aqua regia in ratios of 1:1, 1:5, 1:10. Our results demonstrate that near 100% recovery of Au concentration can be obtained with the appropriate dilutant and dilution ratio. This work facilitates TXRF's transition into AuNP research as it offers an easy-to-use analytical toolbox that can accurately quantify AuNPs in blood.
Lead (Pb) exposure assessment in dried blood spots using Total Reflection X-Ray Fluorescence (TXRF)
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At this stage, the internal standard was added to ensure that it bound to the sample matrix. We evaluated the performance of three internal standards (Ga, Ni, and Sr) previously studied for TXRF-based analysis of Pb (Silva et al., 2013; Stosnach, 2009; Martinez et al., 2004, Bounakhla, 2003), and decided upon the use of Ni at a final concentration of 150 ppb (Supplementary figure S2). As previous studies (including our own pilot efforts) have shown that the relatively high iron levels in blood interfere with TXRF-based detection of some elements (Prangue, 1989; Khunder, 2006), we included Methyl Isobutyl Ketone (MIBK) to the digested samples to extract out the iron (more details on the extraction process are in Supporting information S5).
Lead (Pb) exposure is often determined through the analysis of whole blood though venipuncture poses ethical, economic, and logistical barriers. Dried Blood Spots (DBS) may help overcome such barriers though past studies measuring Pb in DBS have been challenged with quality control, small sample volumes, and other issues. Total Reflection X-Ray Fluorescence (TXRF) may help address some of these challenges but has yet to be used to measure Pb in DBS. As such, the aim of the current study was to develop, validate, and apply a method to analyze Pb in DBS samples using TXRF for use in human biomonitoring studies. First, we developed a novel method (tested a range of parameters), and then used blood reference materials to validate the method against performance criteria listed in ICH Q2A and Q2B and the European Bioanalysis Forum. Finally, we applied the method to two populations who exemplify divergent conditions (41 university members with relatively low Pb exposures sampled in a clinical environment; 40 electronic waste workers with relatively high Pb exposures sampled in a contaminated field setting). The limits of detection and quantification of the method were 0.28 and 0.69 μg/dL, respectively. The overall precision and accuracy of the method were 15% and 111%, respectively. The mean (±SD) DBS Pb levels by TXRF in the university members and e-waste workers were 0.78 (±0.46) and 3.78 (±3.01) μg/dL, respectively, and these were not different from Pb measures in venous whole blood using ICP-MS. Bland-Altman plot analyses indicated good agreement between DBS Pb measures by TXRF versus whole blood Pb measures by ICP-MS in both groups. By combining data from the two population groups, there was no significant constant bias (intercept of 0.02 μg/dL) or proportional bias (slope was −0.02) between the two measures, and the lower and upper LoA were −0.86 and 0.91 μg/dL, respectively, with a LoA range of 1.77 μg/dL. These results demonstrate that TXRF-based analysis of Pb content in DBS is a good alternative to the gold standard (i.e., ICP-MS analysis of whole blood), and helps overcome some of the challenges associated with current methods.
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However, this analysis is quite expensive and requires access to a complex instrumentation, which is not widely available and easy to manage. Total-reflection X-ray spectroscopy (TXRF) is nowadays a well-established technique which is applied in several fields such as environmental science [10–12], geology [13–15], materials science [16], food science [17], medicine [18,19]. The advantages of TXRF over other analytical techniques are the low amount of sample required, fast sample preparation, easy sample pre-treatment, and relatively low costs for the instrumentation and its operation.
Cesiumlead-halide perovskite nanocrystals are an emerging class of materials which potentially have different applications due to the several physical properties they exhibit. These properties are strongly dependent on the elemental composition of the nanocrystals and, to date, only few methods are available for their chemical analysis, such as scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX). The present work aims at establishing a new, fast and simple method for the elemental analysis of cesium lead-halide perovskite nanocrystals exploiting total-reflection x-ray fluorescence (TXRF) spectroscopy. The method was validated using a synthetized set of samples and comparing the TXRF results with SEM-EDX data. The sample preparation consisted in suspending the perovskites in 2.0 ml of hexane and sampling 10 μl of the suspension for deposition on a preheated quartz carrier. The element recovery ranged between 82% and 118% for mixed-halide perovskites, while for single halide perovskites it improved to 86%–105%. The present method can be implemented and used also for the elemental characterization of other types of perovskite nanocrystals.
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Beside multi-elemental analysis that allow analysis of higher number of elements from the same urine sample, TXRF method is simple and not time-consuming [28]. The applied TXRF method was validated as reported previously [11] and for quality control a certified urine reference sample (Seronorm urine level 2, SERO AS, Norway) was used. Additionally, obtained baseline values for urinary Zn, Se, Na, K, Ca and P in the present study were comparable to values reported previously confirming TXRF reliability [2,29–31].
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Earthworms are often used as sentinel organisms to study As bioavailability in polluted soils. Arsenic in earthworms is mainly sequestrated in the coelomic fluids whose As content can therefore be used to asses As bioavalability. In this work, a method for determining As concentration in coelomic fluid extracts using total-reflection X-ray fluorescence spectrometry (TXRF) is presented. For this purpose coelomic fluid extracts from earthworms living in three polluted soils and one non-polluted (control) soil have been collected and analysed. A very simple sample preparation was implemented, consisting of a dilution of the extracts with polyvinyl alcohol (PVA) using a 1:8 ratio and dropwise deposition of the sample on the reflector. A detection limit of 0.2 μg/l and quantification limit of 0.6 μg/l was obtained in the diluted samples, corresponding to 2 μg/l and 6 μg/l in the coelomic fluid extracts, respectively. This allowed to quantify As concentration in coelomic fluids extracted from earthworms living in soils polluted with As at concentrations higher than 20 mg/kg (considered as a pollution threshold for agricultural soils). The TXRF method has been validated by comparison with As concentrations in standards and by analysing the same samples by ICP-MS, after acid digestion of the sample. The low limit of detection, the proven reliability of the method and the little sample preparation make TXRF a suitable, cost-efficient and “green” technique for the analysis of As in earthworm coelomic fluid extracts for bioavailability studies.

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^☆: This paper was presented at the 12th Conference on Total Reflection X-ray Fluorescence Analysis and Related Methods held in Trento (Italy), 18–22 June 2007.

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Analytical noteAnalysis of nutrition-relevant trace elements in human blood and serum by means of total reflection X-ray fluorescence (TXRF) spectroscopy☆

Abstract

Introduction

Section snippets

Instrumentation

Samples

Accuracy

Conclusions

Spectrochim. Acta Part B

Spectrochim. Acta Part B

Spectrochim. Acta Part B

Spectrochim. Acta Part B

Multi-element determination of trace elements in whole blood and serum by TXRF

Fresenius Z. Anal. Chem.

Analytical note
Analysis of nutrition-relevant trace elements in human blood and serum by means of total reflection X-ray fluorescence (TXRF) spectroscopy☆