Analytical potential of total reflection X-ray fluorescence spectrometry for simultaneous determination of iron, copper and zinc in human blood serum and plasma

doi:10.1016/j.talanta.2021.122553

Talanta

Volume 233, 1 October 2021, 122553

https://doi.org/10.1016/j.talanta.2021.122553 Get rights and content

Highlights

•
Simple determination of Fe, Cu and Zn in plasma and serum samples by TXRF.
•
Method evaluation by experimental design tools (surface response analysis).
•
Best strategy: direct analysis of serum and plasma samples without a dilution step.
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Limits of detection: 0.03–0.14 mg L⁻¹ (plasma) and 0.05–0.33 mg L⁻¹ (serum).
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Good agreement between TXRF and ICP-based methods results.

Abstracts

Due to many roles of trace elements such as Fe, Cu and Zn in various physiological and pathophysiological processes, their determination in serum and plasma is of high clinical relevance.

In the present study, for the first time, the effect of serum and plasma preparation parameters (dilution factor and sample deposition volume) on the quality of results obtained by TXRF analysis was evaluated by means of experimental design tools (response surface analysis). It was found that the best strategy was the direct analysis of both human fluids without a previous dilution step.

The accuracy and precision of the proposed methods were evaluated by analysis of reference materials (ClinChek® Plasma Control Level II and Seronorm™ Trace Elements Serum L-1). TXRF results agreed with the reference values and no significant differences at 95% confidence level were found. Limits of detection for the elements of interest were also adequate, taking into account their typical concentration ranges in real serum and plasma samples.

Finally, the developed TXRF methods were applied to a set of serum and plasma samples from patients with different genders, ages and diagnoses, previously analysed by ICP-OES and ICP-MS techniques. The results showed good agreement between both analytical approaches. These results suggest that the proposed TXRF method provides reliable results thus being suitable for plasma and serum analysis, but in a simpler and more sustainable way.

Graphical abstract

Introduction

Due to the important roles of different elements in the physiological processes and their role in usefulness in the diagnostics and prognostics of various diseases as well as in biomonitoring, the determination of major, minor, trace and toxic elements in human body fluids such as whole blood, plasma and serum has become of high scientific and clinical relevance [1,2]. The most used samples for clinical laboratory analyses are serum and plasma. The main difference between these two samples is the presence of fibrinogen in plasma, since sampling is performed in tubes with different anticoagulants (K₂ or K₃-EDTA, lithium heparin, sodium citrate, sodium fluoride …) whereas serum is obtained by venepuncture into the tubes without anticoagulant, thus lacking fibrinogen due to fibrin and clot formation.

According to the WHO guidelines, clinically more relevant samples for electrolytes and trace elements analyses using standard methods are serum and urine than plasma due to anticoagulants used in plasma sample preparation, which are polyanions or metal chelators with high affinity for metal ion binding, or ammonium, lithium, sodium or potassium salts. However, the use of particular sample highly depends on the applied method, and every method should be validated regarding to the sample type and type of coagulant in case of plasma samples usage [3].

Methods used for trace elements determination in serum and plasma samples vary between laboratories but most of them use atomic spectroscopic techniques such as graphite furnace atomic absorption spectrometry (GFAAS) [4], hydride generation atomic absorption spectrometry (HGAAS) [5,6] or inductively coupled plasma-mass spectrometry (ICP-MS) [7,8].

Another less explored analytical possibility is the use of total reflection X-ray fluorescence spectrometry (TXRF). TXRF offers some advantages with respect to other spectroscopic techniques, such as the low amount of sample required, which is of special interest in human biological fluids, simultaneous multielemental capability, and easy quantification by means of internal standardization [9]. Further, low power benchtop TXRF systems are cost-effective comparing to ICP-OES and ICP-MS techniques because gas or cooling media are not required for their functioning. An additional advantage of TXRF is the possibility to analyse complex biological liquid samples using simpler sample treatments that are more in line with the Green Analytical Chemistry principles than other spectroscopic techniques. The overview of publications on the use of TXRF methods for the analysis of serum and plasma samples in the last ten years (2011–2021) is shown in Table 1. As can be seen, serum has been more often used for TXRF analysis than plasma samples.

To perform analysis under total reflection conditions, samples must be provided as thin films. For liquid samples, it is usually done by depositing few μL of the sample on a reflective carrier with subsequent drying of the drop. In the case of biological fluids such as serum and plasma, TXRF analysis can be directly performed by depositing 2–20 μL of either raw or diluted sample (1:1 or 1:2) on a suitable reflector (see Table 1). In only one study, acid digestion was employed for serum sample treatment before TXRF analysis [14]. In fact, more sophisticated sample treatments are required for the analysis of biological fluids with a more complex matrix (i.e., whole blood) or to improve limits of detection, as it has been previously reported [18,19].

Although both serum and plasma can be used for TXRF analyses, see Table 1, there is no consensus on which of these two samples is more suitable for such analysis as well as about the best experimental conditions (in terms of dilution ratio and sample deposition volume) for getting the most reliable analytical results. In fact, most of the aforementioned publications did not provide an evaluation of the sample treatment procedure and, even, the analysis of adequate certified reference materials was not performed. Therefore, it is difficult to evaluate the analytical capabilities of the TXRF method and the quality of the obtained results. In view of these premises, the aim of the present study was to perform a detailed study on the effect of dilution factor and sample deposition volume on the quality of the TXRF results obtained for multielement analysis of serum and plasma samples. For that purpose, a detailed consideration of the variables ranges was intended using a response surface analysis designed in terms of a central composite design [20]. The quality of developed methods was confirmed using the reference materials (ClinChek® Plasma Control Level II and Seronorm™ Trace Elements Serum L-1).Finally, the TXRF methods were applied to a set of serum and plasma samples from patients with different genders, ages and diagnoses, previously analysed by ICP-OES and ICP-MS techniques. As far as the author's knowledge, it is the first study in which careful evaluation of the sample treatment procedure for both serum and plasma was performed by means of experimental design tools. The obtained results provide a good basis for further development of this technique for application in routine clinical laboratories.

Section snippets

Reagents and materials

Monoelemental stock solutions of 1000 mg L⁻¹ (ROMIL PrimAg@ Monocomponent reference solutions) were used for preparing the internal standard (IS) solutions, spiked serum and plasma samples and calibration standards for ICP-MS/ICP-OES analysis. Ultrapure de-ionized water used for dilution of stock solutions and samples was obtained by a Milli-Q purifier system (Millipore Corp., Bedford, Massachusetts). A solution of 1% of Triton™ X-100 in water (laboratory grade, Sigma-Aldrich) and a 0.2 M HNO₃

Optimization of TXRF analysis of serum and plasma samples

In the first part of this study, preliminary tests to evaluate sample deposition on the reflector, in terms of shape, size and stability were performed. For that purpose, aliquots of serum and plasma samples (10 μL) were deposited on quartz reflectors with or without a previous dilution step. Different diluting agents were tested: ultrapure water, solution of 1% Triton X-100 and 0.2 M nitric acid. Higher concentrations of nitric acid were not applied because of the precipitation of proteins

Conclusions

This study provide insight into the effects of most relevant sample treatment parameters on the quality of the results of Fe, Cu and Zn concentration measurements in plasma and serum samples by TXRF with W X-ray tube. According to the results obtained by means of an experimental design tool, better results were obtained by direct analysis of plasma and serum samples, without a dilution step. This is a clear advantage of TXRF method in comparison with atomic spectroscopy and ICP-OES/ICP-MS

Credit author statement

J.Jablan: Conceptualization, Methodology, Validation, Investigation Resources, Writing – original draft, Writing – review & editing, Visualization, Funding acquisition. E.Besalú; Methodology, Software, Formal analysis, Investigation Resources, Writing – review & editing, Visualization. M. Žarak: Conceptualization, Investigation Resources, Writing – review & editing. J. Dumić; : Conceptualization, Investigation Resources, Writing – review & editing. E.Marguí; : Conceptualization, Methodology,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

“This article/publication is based upon work from COST Action CA18130 ENFORCE TXRF, supported by COST (European Cooperation in Science and Technology).” COST (European Cooperation in Science and Technology) is a funding agency for research and innovation networks. Our Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation (http://www.cost.eu/).

References (32)

P. Parsons et al.
Atomic spectrometry and trends in clinical laboratory medicine
Spectrochim. Acta Part B-Atomic Spectroscopy - SPECTROCHIM ACTA PT B-AT SPEC.
(2007)
M. Gallignani et al.
Sequential determination of Se(IV) and Se(VI) by flow injection-hydride generation-atomic absorption spectrometry with HCl/HBr microwave aided pre-reduction of Se(VI) to Se(IV)
Talanta
(2000)
M.G.C. Fernandez et al.
Flow-injection and continuous-flow systems for the determination of Se(IV) and Se(VI) by hydride generation atomic absorption spectrometry with on-line prereduction of Se(VI) to Se(IV)
Anal. Chim. Acta
(1993)
M. Krachler et al.
The potential of inductively coupled plasma mass spectrometry (ICP-MS) for the simultaneous determination of trace elements in whole blood, plasma and serum
J. Trace Elem. Med. Biol.
(1999)
S. Griesel et al.
Mineral elements and essential trace elements in blood of seals of the North Sea measured by total-reflection X-ray fluorescence analysis
Spectrochim. Acta B Atom Spectrosc.
(2006)
E.D. Greaves et al.
Rapid determination of platinum plasma concentrations of chemotherapy patients using total reflection X-ray fluorescence
Spectrochim. Acta B Atom Spectrosc.
(2006)
N. Szoboszlai et al.
Recent trends in total reflection X-ray fluorescence spectrometry for biological applications
Anal. Chim. Acta
(2009)
A. Stojsavljević et al.
Determination of toxic and essential trace elements in serum of healthy and hypothyroid respondents by ICP-MS: a chemometric approach for discrimination of hypothyroidism
J. Trace Elem. Med. Biol.
(2018)
R. Dalipi et al.
Analytical performance of benchtop total reflection X-ray fluorescence instrumentation for multielemental analysis of wine samples
Spectrochim. Acta B Atom Spectrosc.
(2016)
J.L. Luque-Garcia et al.
Sample preparation for serum/plasma profiling and biomarker identification by mass spectrometry
J. Chromatogr. A
(2007)

Ch Zarkadas et al.

Applicability of direct total reflection X-ray fluorescence analysis in the case of human blood serum samples

Spectrochim. Acta B Atom Spectrosc.

(2001)

M.L. Bishop et al.

Clinical Chemistry: Principles, Techniques, and Correlations

(2013)

H.G. Seiler et al.

Handbook on Metals in Clinical and Analytical Chemistry

(1994)

Use of Anticoagulants in Diagnostic Laboratory Investigations

(2002)

N. Laur et al.

ICP-MS trace element analysis in serum and whole blood

PloS One

(2020)

E. Margui et al.

X-Ray Fluorescence Spectrometry and Related Techniques: an Introduction

(2013)

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There are usually many methods to determine copper such as high- inductively coupled plasma, atomic fluorescence spectrometry, X-ray fluorescence spectrometry and electrochemical methods. Most of these methods are very expensive and time consuming, whereas the electrochemical methods have high sensitivity, simplicity, and rapid response [9–13,6]. In the present study, we used a simple and inexpensive way for the size-controlled synthesis of cubic shaped AF-meso-Fe3O4NPs.
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Analytical potential of total reflection X-ray fluorescence spectrometry for simultaneous determination of iron, copper and zinc in human blood serum and plasma

Highlights

Abstracts

Graphical abstract

Introduction

Section snippets

Reagents and materials

Optimization of TXRF analysis of serum and plasma samples

Conclusions

Credit author statement

Declaration of competing interest

Acknowledgments

Spectrochim. Acta Part B-Atomic Spectroscopy - SPECTROCHIM ACTA PT B-AT SPEC.

Talanta

Anal. Chim. Acta

J. Trace Elem. Med. Biol.

Spectrochim. Acta B Atom Spectrosc.

Spectrochim. Acta B Atom Spectrosc.

Anal. Chim. Acta

J. Trace Elem. Med. Biol.

Spectrochim. Acta B Atom Spectrosc.

J. Chromatogr. A

Spectrochim. Acta B Atom Spectrosc.

Clinical Chemistry: Principles, Techniques, and Correlations

Handbook on Metals in Clinical and Analytical Chemistry

Use of Anticoagulants in Diagnostic Laboratory Investigations

ICP-MS trace element analysis in serum and whole blood

PloS One

X-Ray Fluorescence Spectrometry and Related Techniques: an Introduction