Rare earth elements (REEs) are widely used in areas of agriculture, national defense, new energy, biological medicine, aerospace and the nuclear industry and daily life [1
], such as fertilizers, automotive catalysts, luminescent materials, high-performance permanent magnets, contrast agents in biomedical imaging, antitumor medicine, nuclear radiation detector [3
]. Wide utilization implies the current- and growing-spread of REEs in environmental and occupational exposure. The literature from animal studies and limited data from human occupational exposures suggest that REEs have redox reactivity, involving ROS formation, lipid peroxidation and modulation of antioxidant activities, have ephro- and hepato-toxicity, and can induce tissue-specific bioaccumulation [5
To assess the potential risk to human health, it is necessary to investigate the exposure level of REEs, namely “source emissions environmental concentration-exposure human biological monitoring- health effects surveillance”. In this continuum, biological monitoring is an accurate and reliable complement to environmental monitoring [9
]. Biological monitoring of exposure integrates the absorption incurred from all sources and routes of exposure [13
]. Metal levels in biological specimens (sputum, blood, urine, hair, nails, etc.
) can reflect the total exposure from all possible sources based on some reports [4
]. Compared with other biological specimen, urine is commonly used for the direct analysis due to its less invasive, easily available, simple mode of collection, storage and sample preparation [18
]. Urine is not only an excretory medium, but also a biological sample for assessment of renal functions [19
]. The urinary REEs can be quantified quickly and have been widely used to directly reflect the recent human environmental exposure [5
In order to monitor the levels of the REE(s) in various tissue fluids, the effective methods for sample preparation and determination are necessary [23
]. Currently, the techniques for simultaneous determination of multiple trace elements in human body mainly include inductively coupled plasma atomic emission spectroscopy (ICP-AES), neutron activation analysis, isotope dilution thermal ionization mass spectrometry (ID-TIMS). These techniques have made a marked improvement in the sensitivity, but their detection limits are still unsatisfactory. In 1983, inductively coupled plasma mass spectrometry (ICP-MS) was introduced as a commercially available system with great progress and currently used for a wide range of applications [26
]. Today, ICP-MS has become one of the most effective techniques for simultaneous determination of multiple trace or ultra-trace elements (e.g., REEs) in human biological samples, high-purity materials, and geological samples [1
]. Among several analytical techniques used to determine the concentration of REEs in urine, ICP-MS technology has the rapid, quasi-simultaneous, multi-element detection capabilities, low detection limits and high sensitivity. It has been used in the quantitative analysis of the individual elements, qualitative and semi-quantitative analysis of all the elements present, and analysis of isotopic ratios [20
]. However, the oxide/hydroxide ions formed by light REEs can affect the ICP-MS determination of heavy REEs. Thus, more attention should be focused on the spectral interferences and matrix effects. Spectral interferences occur when two or more molecular or atomic species have the same nominal mass-to-charge ratio so the signal at that mass cannot be resolved [38
]. Unlike spectral interferences, matrix effects can not only overlap or enhance the signal, but also cause many physical/chemical effects [39
]. Therefore, in some complex samples, a number of unexpected interferences may arise, confusing spectra and increasing the risk of erroneous quantification [41
Human urine contains a high proportion of total dissolved solids (TDS) and salt, the TDS may lead to signal suppression and salts often build up on the cones and torch of the ICP-MS instrument after introduction of even a few milliliters of sample [23
]. Therefore, matrix simplification of urine samples by dilution and/or digestion is often required before analysis to reduce the effects of polyatomic interferences, matrix-induced signal suppression and carbon-enhanced ionization effects in the plasma. Traditional methods (e.g., sample digestion) require extensive sample preparation, which may increase the chance of contamination or loss of sample, thus increasing experimental uncertainty.
The primary aim of this work was to explore a sensitive and reliable indicator of exposure level to rare earth elements. This tool could be used to enhance the health risk assessment and management of workers manufacturing cerium, lanthanum oxide ultrafine and nanoparticles. In this study, an ICP-MS method for quantification of 15 REEs (Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu) concentrations in diluted human urine was developed and validated. This method was then applied for the determination of urinary samples obtained from 8 control subjects and 23 workers that manufacture cerium and lanthanum oxide ultrafine and nanoparticles.
2. Materials and Methods
The urine samples were collected in metal-free polypropylene containers and stored at −20 °C. Prior to sample collection, the time of sampling and working hours were collected for the biological monitoring protocol. The exposed subjects investigated (n = 23) were the workers employed in a professional enterprise engaged in the manufacture and sale of rare earth powder products. The primary products are cerium, lanthanum oxide ultrafine and nanoparticles—the particle diameters ranged from 0.05 to 0.8 μm. The control subjects investigated (n = 8) were the support staff and management personnel from the same enterprise. All of the subjects (n = 31) were informed that their urine would be used for REEs determination and agreed to participate in this study. This project financial supported by University of Macau Research Grant, and the project has been approved by Ethical Committee of the University Board, code number “MYRG106 (Y1-L3)-ICMS13-BY”.
Nitric acid (Trace SELECT® Ultra) was purchased from Sigma Chemicals Ltd. (St. Louis, MO, USA). The rare earth elements standard solution containing Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu (100 mg∙L−1 for each) was purchased from Shanghai Institute of Quality Inspection and Technical Research (Shanghai, China). Standard Tune B iCAP Q solution contaning Ba, Bi, Ce, Co, In, Li, and U (1.0 μg∙mL−1 each) was purchased from Thermo Fisher Scientific (Bremen, Germany). Water with a resistivity of 18.2 M∙cm−1 was prepared using a Milli-Q system (Millipore, S.A., St. Quentin Yvelynes, France) and used throughout this work.
Rare earth element determination was performed by an iCAP™ Q ICP-MS (Thermo Fisher Scientific, Bremen, Germany), typical operating parameters are given in Table 1
. Urine samples were introduced by an auto sampler CETAC ASX-520 (CETAC Technologies, Inc., Omaha, NE, USA). Tuning was performed daily using the standard auto tune parameters. Data acquirement and analysis were performed with the software of Qtegra™ Intelligent Scientific Data Solution™ (Qtegra, version 2.4.1800.192).
2.4. Sample Preparation and Quality Control
Matrix-matched calibration curves are widely used for the analysis of biological samples to account for matrix effects in inorganic mass spectrometry. In this study, a diluted base urine sample (20-fold dilution with 2% HNO3) was used for matrix matching the calibration standards, which ranged from 0.001 to 1.000 μg∙L−1. The elements with no isobaric interferences were determined. The monitored elements were 89Y, 139La, 140Ce, 141Pr, 146Nd, 147Sm, 153Eu, 157Gd, 159Tb, 163Dy, 165Ho, 166Er, 169Tm, 172Yb and 175Lu in standard mode. Calibration curve for each element was constructed by plotting the concentration of individual element as a function of signal intensity.
For quality assurance and control, blank spikes (0.01, 0.1 and 1.0 μg∙L−1 for all REEs) and standard solution (QC, 0.1 μg∙L−1 for all REEs) were used during analyses. The intra-day (CV% (1)) and inter-day (CV% (2)) precisions were defined as the relative standard deviation (RSD) of six replicates of QC sample within 1 day or the QC sample on five separate days, respectively. The recovery was estimated by comparing the determined concentrations of individual element with that of spiked concentrations in samples. The limits of detection (LOD) and limit of quantification (LOQ) for REEs were determined as three times and 10 times of the standard deviation from 11 independent analyses of the base urine. The human urine samples were diluted 20-fold with 2% HNO3 before ICP-MS analysis.
2.5. Statistical Analysis
All experiments were performed in triplicate. The data were analyzed with IBM SPSS Statistics 22.0 software package. The concentration of 15 REEs are presented as mean ± S.D, median, range and 25th–75th percentile. Values under the LOD were substituted with half of LOD in the computation of means [43
]. Variance between the control subjects and the exposed workers was evaluated by Student’s t
-test and one-way analysis of variance (one-way ANOVA), respectively. A value of p
< 0.05 was considered significant for all tests.