HPLC-ICP-MS coupling for mercury speciation in seafood. A performance comparison between MassNeb® and Micromist® nebulizers

1. Introduction

Mercury is an element of undisputed significance, as it is considered one of the most toxic elements in the periodic table.
The toxicity of mercury not only depends on its concentration but also on the chemical form in which it is present. It is well known that alkylated species of mercury are much more toxic than inorganic or elemental mercury because they can easily cross cellular membranes and accumulate with high efficiency in organisms. Additionally, alkylmercury is not well metabolized and tends to be bioaccumulated and biomagnified, increasing its concentration in top predators in the food chain. For this reason, speciation analysis is increasingly important in routine applications, particularly for samples such as seafood, where safety is a concern.

ICP-MS is widely used to quantify a wide range of elements, including total Hg, in seafood. However, total elemental concentrations do not provide adequate information for food safety assessment when a metal’s toxicity depends on its chemical form. In this sense, ICP-MS can provide speciation analysis of individual Hg compounds, including inorganic mercury (Hg2+) and methylmercury (MeHg), by coupling the ICP-MS to a high-performance liquid chromatography (HPLC) system.

On the other hand, due to the low detection limits required for the analysis of Hg species, GC-ICP-MS has been the preferred method. However, with sensitivity improvements in modern ICP-MS systems, the use of HPLC has become practical for the separation, and derivatization steps for GC separation are not required.

Furthermore, for speciation analysis using the HPLCICP-MS analytical approach, Ingeniatrics Tecnologias S.L. has released a specific one-piece connector for OneNeb® or MassNeb® (one liquid inlet) with a standard length of 500 mm (longer at request) PEEK capillary to connect the exit of the chromatographic column directly to the nebulizer (using high and low pressure, red and green PEEK capillary, respectively).
This replaces the conventional HPLC-ICP-MS connection kit used for this purpose, allowing for a quick, reliable, and easy connection of your HPLC to your ICP-MS. In this study, we evaluate the performance of the MassNeb® inert, robustness, and durability nebulizer, along with a specific connector for speciation analysis designed by Ingeniatrics Tecnologías S.L. for analytical methodologies based on the use of HPLC-ICP-MS for speciation analysis.
HPLC-ICP-MS coupling

2. Experimental

Reagents and solutions

All reagents used for sample preparation were of the highest available purity. Hydrochloric acid (30%) used for the preparation of extractants was of Suprapur® grade (Merck, Darmstadt, Germany). Other commonly used chemicals included methanol (99.9%), Lichrosolv® grade (Merck), 2-mercaptoethanol (2-ME) ≥ 99% and L-cysteine·HCl puriss. p.a. (both Fluka, Buchs, Germany).
Method development and validation were carried out with BCR-463 Tuna Fish (IRMM, Belgium), DOLT-4 dogfish liver and TORT-3 lobster hepatopancreas (National Research Council Canada), three seafood certified reference materials for methylmercury and total mercury contents. The mobile phase (0.1% Lcysteine, pH adjusted to 2.6 using 1.0 M ultrapure grade hydrochloric acid) was used to prepare the calibration standards and food samples.
The calibration blank and standards were prepared in the mobile phase. The inorganic standards were prepared from a stock 10 ppm mercury standard.
Methylmercury stock standard was prepared by dissolving methylmercury chloride (Sigma-Aldrich, Malaysia) in deionized water and adding 2- mercaptoethanol (Merck, Singapore) until methylmercury was the primary Hg species.
Thereafter, calibration standards (0.5, 1, 2, 5 and 10 ppb) were prepared in the mobile phase.


Reverse phase chromatography analysis was performed using an Agilent 1260 HPLC system consisting of a quaternary pump, an autosampler, and a vacuum degasser, which was coupled to an Agilent 7900 ICP-MS. Arsenic species were separated using a Zorbax C-18 column with a size of 4.6 x 50 mm and particle size of 1.8 μm from Agilent Technologies.
Samples and standard solutions were injected through a 100 µL sample loop. The column was maintained at a temperature of 30°C during the chromatographic separation. Mercury compounds were separated using an isocratic elution of 0.1% (w/v) L-Cysteine HCl (98%) and Methanol (2%). The mobile phase flow rate was kept constant at 900 µL min−1 during the chromatographic separation, and the total time for separating the mercury species was 5 minutes.
The Agilent 7900 ICP-MS (Agilent Technologies) provides sensitive and specific analysis of Hg isotopes. The optimization of ICP-MS was carried out using a tuning solution consisting of Cs (cesium, 55), Co (cobalt, 27), Li (lithium, 3), Mg (magnesium, 12), Tl (thallium, 205), and Y (yttrium, 89) (Agilent Technologies, Palo Alto, CA, USA). In this study, MassNeb® nebulizer is proposed as an appropriate nebulizer in speciation analysis based on the use of HPLC-ICP-MS instrument configuration using the OnePiece High Pressure for Speciation Analysis by HPLCICP (Figure 1) (Part Number: CN253005, Ingeniatrics Tecnologías S.L.).
On the other hand, the Agilent LC connection kit for speciation analysis by HPLC-ICP-MS (Part No: G1833-65200) was used with the Micromist nebulizer.
HPLC-ICP-MS coupling

Figure 1. High pressure connector ON / MS speciation 0,125 mm for HPLC-ICP-MS speciation analysis.

Operating conditions for total element determination using ICP-MS by internal standard calibration and speciation analysis by reverse phase chromatography (RP) combined with inductively coupled plasma mass spectrometry (ICP-MS) for mercury species quantification optimized are indicated in Table I.
HPLC-ICP-MS coupling

Table II. Operational conditions for 7900 ICP-MS and 1260 HPLC optimized for mercury speciation by HPLCICP-MS (Agilent Technologies).

Sample Preparation

For determination of total Hg content in all samples studied, approximately 0.2 g was microwave digested in 3.0 mL concentrated HNO3 and 1.5 mL of 30 % w/w H2O2 in XP1500 vessels in CEM Mars microwave system. For total Hg determination, gold was added to the samples and the rinse solution to minimize memory effects from the sample introduction system. The samples were diluted to the final volume of 25 mL with deionized water. Each sample was prepared in triplicate. The resulting solution was filtered through Iso-Disc poly(vinylidene difluoride) filters (25 mm diameter, 0.45 µm poresize). The total content of mercury was measured by ICP-MS using as internal standard rhenium( Re) to 1 µg L-1. A blank with the reagents used for total mercury determination was run simultaneously to the sample preparation. On the other hand, for speciation analysis, he sample extraction and cleanup procedures constitute a crucial step when biota samples are considered, due to possible analyte losses, changes of the species or incomplete extraction of the mercury compounds, which may lead to poor or erroneous results.
For mercury species extraction from the seafood samples (0.2 g) was weighed into the 50.0-mL glass vials and 20 mL of 0.1% (w/v) L-Cysteine HCl (98%) + Methanol (2%) was added. The capped vials were placed in a Digiprep® digestor at 60 °C for 120 minutes. Then, samples were centrifuged, and filtered prior to analysis by HPLC-ICP-MS. All the filters were cleaned with 5 mL of the extracting solution before the use to avoid the contamination of the samples. Post-digestion spikes of 100 ng kg-1 and 1 µg kg-1 Hg were prepared to check spike recoveries of Hg and evaluate whether the presence of a high concentration of inorganic Hg would have any impact on the recoveries of methylmercury during the chromatographic separation.

3. Results and discussion

Chromatographic optimization

For mercury speciation analysis, many studies have used HPLC-ICP-MS as an effective method. Three certified reference materials (CRMs) were used to validate the method. No interconversion among mercury species was observed under the detailed experimental conditions. In addition, mercury species were successfully speciated using previously described operational and experimental conditions in less than 4 minutes. The resulting chromatogram is shown in Figure 2.

Figure 2. High pressure connector ON / MS speciation 0,125 mm for HPLC-ICP-MS speciation analysis.

Sensitivity and Signal stability

MassNeb® nebulizer uses Flow Blurring nebulization technology instead of the traditional Venturi effect, as Micromist® nebulizer. This allows for 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 (Figure 3).

Figure 3. Comparison of size distribution droplet generated by MassNeb and Micromist nebulizer

The optimal aerosol generated by the MassNeb® nebulizer is also more efficiently desolvated and excited in plasma, helping to improve precision values, typically less than 1% RSD between replicates of the same analysis run, even at low sample flow rates, which also explains why it is much more sensitive than conventional nebulizers. Related to this, Figure 4 shows the chromatographic profile obtained for 100 ng L-1 for each mercury species employing both nebulizers. 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.
HPLC-ICP-MS coupling
Table II. Experimental and certified mean values for total mercury and mercury species concentrations in the different seafoods analyzed, as well as the RSD obtained for three replicates by HPLC-ICP-MS.

Figure 4. Chromatographic profile obtained for 100 ng L-1 (as Hg) for each mercury species employing MassNeb® and Micromist® nebulizers.

As can be seen in Figure 4, the results obtained in this study using MassNeb® in combination with the One-Piece High Pressure for Speciation Analysis by HPLC-ICP (Figure 1) (Part Number: CN253005, Ingeniatrics Tecnologías S.L.). provides better sensitivity in comparison with the results when Micromist® nebulizer is employed. In addition, baseline smoothing, and reproducibility is observed using MassNeb® in combination with the One-Piece High Pressure Connector (Figure 5).

Figure 5. Baseline profile obtained for 201Hg by HPLCICP-MS employing MassNeb® and Micromist® nebulizers.

To evaluate the signal stability throughout the analysis sequence, a monitoring standard solution with 100 ng kg-1 of inorganic mercury and methylmercury was checked. This solution was analyzed once every five samples to assess the signal stability. The recoveries were required to be within the range of 94-108%.

Precision and reproducibility evaluation

Precision values were evaluated using certified reference materials (CRMs) of seafood matrix including BCR-463 Tuna Fish (IRMM, Belgium), DOLT-4 dogfish liver, and TORT-3 lobster hepatopancreas (National Research Council Canada), after following the sample treatment previously described. Table II summarizes the experimental registered values for each mercury species obtained using both nebulizers, along with the RSD obtained for 3 replicates of each of the CRMs used in the experimental development of this application note. Precision is expressed as the relative standard deviation percentage (RSD%). In general, MassNeb® showed better precision and reproducibility results compared to Micromist® nebulizer. The increased precision in the results using MassNeb® nebulizer is related to the higher sensitivity and reproducibility reported by the results obtained in comparison with the Micromist® nebulizer.

Furthermore, in order to validate the method performance in real samples, a spike recovery test was performed using the mixed Hg species standard solution. Post-digestion spikes of 100 ng kg-1 and 1 µg kg-1 Hg were prepared to check spike recoveries of Hg and also evaluate whether the presence of a high concentration of inorganic Hg would have any impact on the recoveries of methylmercury during the chromatographic separation. The recovery obtained were 96 – 104 % for MassNeb® nebulizer and 91 – 108 % for Micromist® nebulizer.

4. Conclusions

In this study, it has been demonstrated that the new MassNeb® nebulizer presents higher precision, sensitivity, signal stability and reproducibility in total mercury determination using ICP-MS and mercury speciation quantification based on HPLC-ICP-MS using the One-Piece High Pressure for Speciation Analysis by HPLC-ICP (Figure 1) (Part Number: CN253005, Ingeniatrics Tecnologías S.L.).

Related to this, the high-pressure connector designed by Ingeniatrics Tecnologias S.L. present some advantages, such as resistance to blockage, fast washout, minimized dead volume, and reduced peak broadening. Moreover, the connector is userfriendly and allows for easy and reliable connection of HPLC to ICP instruments at a low cost.

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