Ingeniatrics

ICP-MS trace metal analysis in human urine. A performance comparison between MultiNeb®, MassNeb® and Micromist® nebulizers

1. Introduction

Urine is the most commonly used biological fluid for biomonitoring studies as the method of sample collection is relatively simple and a variety of elements are excreted in urine as their original form or metabolites. Accurate quantification of trace elements in urine by ICP-MS is, however, often challenging due to analyte concentrations at trace and ultratrace levels and elevated levels of background constituents that may add up to 40 g L-1 in typical human urine. In this sense, matrix elements including Cl (1.9–8.4 g L-1), Na (1.2–4.4 g L-1), and K (0.8–2.6 g L-1) are closely associated with spectral and/or non-spectral interferences in ICP-MS analysis, causing signal suppression by inorganic urine components.

The most common approach to reducing matrix effects is the dilution of urine specimens with an acidic or basic solution prior to analysis along with the use of internal standardization. On the other hand, digestion procedures were occasionally employed with oxidizing reagents such as concentrated nitric acid or a mixture of nitric acid and hydrogen peroxide. Additionally, a synthetic-matrix for human urine solution was used for diluting stock standards containing 2% (w/v) NH4OH, 0.1% (w/v) EDTA, and 1% (w/v) NaCl. Alternatively, organic solvents such as methanol have been widely used in ICP-MS analysis. The direct addition of this solvent to the nebulizer or spray chamber has demonstrated a significant improvement in instrumental sensitivity.

Chromium speciation in waste waters with Massneb and Multineb

Here we describe a simple, quick and robust method for the sequential measurement and quantification of the trace elements As, Ba, Be, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Pb, Sb, Se, Ni, Tl, Pt, V and Zn in human urine using a simple sample preparation method for a small volume of sample and ICP-MS instrument equipped with collision/reaction cell was used to reduce polyatomic interferences and internal standards were used to counter the matrix effects and instrument drift. Conventionally, the internal standard is mixed with the calibration standards and samples using a Y connection, when MassNeb® (Ingeniatrics Tecnologías S.L.) or Micromist® (GlassExpansion) nebulizers are employed. However, the novel MultiNeb® (Ingeniatrics Tecnologías S.L.) has been developed which allows a high mixing efficiency between two liquids, miscible or immiscible, since the mixing takes place under turbulent conditions of high pressure at the tip of the nebulizer. In this work, we compare the performance  of MultiNeb®, MassNeb® and Micromist® nebulizers for simultaneous determination of elements ranging from mg Kg−1 to ng Kg−1 in human  urine by ICP-MS

2. Experimental

Reagents and solutions

All reagents used were of the highest available purity. Methanol (99.9%), Lichrosolv® grade (Merck, Darmstadt, Germany) was employing as organic solvent causing adverse polyatomic interferences on analyte background signals. Helium was used as collision gas, respectively, in an ICP-MS system, were of high-purity grade (>99.999%). Water was purified with a Milli-Q Gradient system (Millipore, Watford, UK). The aqueous levels of calibration standards of 0.005, 0.01, 0.05, 0.10, 0.25, 0.50, 1, 5, 10, 50 and 100 µg Kg-1 were prepared by appropriate dilution of a Multi-element calibration standard-2A (Agilent Technologies, Part Number: 8500-6940). All aqueous solutions are acidified by adding 5% (v/v) high-purity nitric acid (Suprapur®, Merck, Darmstadt, Germany). As internal standard, a mixture of Iridium (Ir), Rhenium (Re) and Rhodium (Rh) in a 1 µg L−1  concentration was employed. High purity NH4OH, EDTA, and NaCl (Aldrich, 99.999% trace metals basis) as a matrix-matching component were used. For comparative analysis of the trace element concentrations in human urine, a serial dilution of the reference materials were used for lyophilized Clinchek human serum Level I and Level II (Recipe Chemicals, Munich, Germany).

Determination of Hg by ICP-MS can be affected by its infamous memory effect in the sample introduction system (tubing, nebulizer, spray chamber) and some instrumental components such as lens, collision cell and quadrupole. When a washout time between runs is not long enough, analytical accuracy and precision can deteriorate, or a calibration curve may become non-linear. For this purpose, 25 µg Kg-1 of gold in a 5% (v/v) HNO3 was employed as a rinse solution.

Instrumentation

An Agilent 7900 ICP-MS (Agilent Technologies, Tokyo, Japan) equipped with standard nickel cones, connected to an SPS 4 Autosampler (Agilent Technologies, Tokyo, Japan), was used for all measurements. equipped with a SPS4 autosampler (Agilent Technologies) with 0.5 mm ID sampling probe (Agilent Technologies, Part No.:G8410-80101).

All measurements were performed in triplicates from each vial. The instrument parameters are described in Table 1. Full data were recorded with the Agilent MassHunter Data software (version 4.2).

ICP-MS Operational Conditions

Table I. Operational conditions for 7900 ICP-MS using online internal calibration (Agilent Technologies).

Conventionally, for multielement quantification by internal calibration, the internal standard is mixed with the calibration standards and samples using a Y connection, using conventional nebulizers, such as MassNeb® and Micromist®. Recently, the novel MultiNeb® has been developed which allows a high mixing efficiency between two liquids, miscible or immiscible, since the mixing takes place under turbulent conditions of high pressure at the tip of the nebulizer. Additionally, in this study the internal solution is composed by a 5% (v/v) of MeOH, 5% (v/v) HNO3 and 1 µg L-1 of a mixture to Rh, Y and Re as internal standards (Figure 1).

Matrix-matched calibration and QC samples

As a previously mentioned, in this study, different levels of calibrations were prepared with a basic solution containing 2% (w/v) NH4OH, 0.1% (w/v) EDTA, and 1.0 % (w/v) NaCl to matrix-match urine specimens. Additionally, For the study of signal stability and plasma drift for multielement determination in human urine, a monitoring standard solution as quality control (QC) containing appropriated concentrations of each elements was prepared. This solution was analyzed once every 5 CRM samples, in order to evaluate the stability of the signal.

Sample preparation

ClinChek® urine controls are used in our study for internal quality assurance. These lyophilized controls are based on calf serum and are available with mean values in the therapeutical as well as in the elevated range. For the reconstitution, 10.0 mL of MilliQ water were added to the vial and was mixed for 30 min. When all material is dissolved, the solution is ready to use.

For multielement determination by ICP-MS 2.5 mL of filtered human urine samples, blanks, calibration levels QC samples were incubated 60 min at 120 ºC after 1 mL of HNO3 (65% (w/v)) addition in a capped glass vial to 10 mL by heating using a heating block system type DigiPREP MS (SCP Science, Courtaboeuf, France) (Figure 2). Then, resulting solutions were diluted three times with a solution containing 2% (w/v) NH4OH, 0.1% (w/v) EDTA, and 1.0 % (w/v) NaCl and filtered through Iso-Disc poly (vinylidene difluoride) filters (25 mm diameter, 0.45 µm pore-size) prior to analysis by ICP-MS.

Figure 2. DigiPREP MS (SCP Science, Courtaboeuf, France).

Schematic representation of MultiNeb and Massneb

Figure 1. A) Schematic representation of MultiNeb®-based configuration for total element determination by internal standard calibration technique by ICP-MS.

B) Schematic representation of MassNeb® or Micromist® -based configuration for total element determination by internal standard calibration technique by ICP-MS.

3. Results

Sensitivity and signal stability

Nebulizers designed by Ingeniatrics Tecnologías SL, such as MassNeb® and MultiNeb®, use Flow Blurring nebulization technology instead of the traditional Venturi effect, as the Micromist® nebulizer does. This allows the generation of a very fine droplet aerosol with a narrow size distribution (most droplets are smaller than 10 μm), which improves efficiency over a wide range of nebulization gas flow rates, especially 0.60-0.75 L min-1 (150-250 kPa nebulization pressure).

Related to this fact, the optimal aerosol generated by the MassNeb® or MultiNeb® nebulizers is also more efficiently desolvated and excited in plasma.

Additionally, the method detection limits (MDLs) were established by analyzing five replicate injections of the calibration blank and multiplying the obtained standard deviation by three. The results obtained are show in Table II.

Precision and reproducibility

Precision values were evaluated using different certified reference materials (CRM) following the procedures for sample preparation previously described. The results obtained are shown in Table II.

For the study of signal stability and plasma drift, a monitoring standard solution as quality control (QC) was analyzed once every 5 CRM samples, in order to evaluate the stability of the signal. The recoveries must fall within the limits of 95-106 %, 91-107% and 96-104 % using MassNeb®, Micromist® and MultiNeb® nebulizers, respectively.

Experimental and certified values using Massneb and Multineb

Table II. Experimental and certified values for each isotope monitored, LOD as well as the RSD obtained for 10 replicates of the different CRM using Micromist® , MassNeb® and MultiNeb® nebulizers employed in this work.

Generally, as can be seen in Table II, the results obtained in this study using MultiNeb® nebulizer provides better precision, sensitivity and reproducibility. In this sense, the highest reproducibility is also observed in the intensity of the internal standards used in this study, as shown in Figure 3 for 185Re isotope. This fact demonstrates that the mixing of the internal standard and the samples/standards solutions is much more efficient and complete in the MultiNeb® than at a Y connection is employed.

On the other hand, it is demonstrable the highest tolerance to TDS using MultiNeb® nebulizer for multielement analysis by ICP-MS in human urine samples. Additionally, we can observe in Table II the higher precision using MultiNeb® nebulizer in comparisons with MassNeb® or Micromist® nebulizers. Related to this, in Figure 3 we can observe, as example, the higher precision in calibration curve obtain using MultiNeb® nebulizer for three of most important elements for clinical and epidemiological studies, such as As, Hg and Cd (Figure 4).

Ratio intensity Massneb

Figure 3. Ratio Intensity evolution to 185Re Intensity as internal standard using MassNeb, Micromist and MultiNeb nebulizers.

Calibration curves for As, Hg and Cd using MultiNeb

4. Conclusions

The results obtained in this study using MultiNeb® nebulizer provides better precision, sensitivity and reproducibility for multielement determination infiltered human urine by ICP-MS in comparison with the results when Micromist® or MassNeb®  nebulizers are employed for this purpose. The same internal diameter is used inside the internal capillaries.

On the other hand, the improved precision results obtained with the MultiNeb® nebulizer are associated with the higher sensitivity and reproducibility achieved when compared to the Y-joint MassNeb® or Micromist® nebulizers. This demonstrates that the mixing of the internal standard and the calibration standard is much more efficient and complete in the MultiNeb® than at a Y-connection.

The novel MultiNeb® enables high mixing efficiency between two liquids, whether they are miscible or immiscible, as the mixing occurs under high-pressure turbulent conditions at the nebulizer’s tip, precisely at the moment when the aerosol is generated. This minimizes the impact on the nebulization process and, consequently, enhances the analytical operation and results.

Finally, this work has demonstrated that the MultiNeb® nebulizer has a higher tolerance for total dissolved solids (TDS). This characteristic makes the MultiNeb® nebulizer more suitable for the analysis of matrices with a high TDS content, such as seawater.

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