Performance comparison in selenoproteins quantification by 2D-LCSE-AF-ICP-QqQ-MS in human serum using two different nebulizers: MultiNeb® and MicroMist®

1. Introduction

It is well known that selenium (Se) is an essential element in humans, and the importance of its biochemistry has been widely reported. This element is present into various proteins, which have many biological functions related to its essential role in human health.

The selenium species most abundant in the bloodstream is the SeP, and its concentration is a good indicator of Se status in humans and antioxidant activity, while eGPx activity in human serum is a complementary marker of selenium status in several clinical studies. Both Se-proteins are interrelated, because Se bound to albumin is assumed to be transported to the liver for new synthesis of SeP that is then released into the bloodstream [1]. In addition, selenometabolites in the bloodstream is readily taken up by red blood cells (RBCs) and reduced to selenide (SeH- ), for latter effluxion into the bloodstream in the presence of albumin and transferred to the liver in the form of SeAlb for the synthesis of selenoproteins [2].

For these reasons, a good approximation of the selenium balance can be obtained by the quantification of eGPx, SeAlb, SeP, selenometabolites in human serum. For this purpose, in this Application Note, a method for the quantification of seleniumtagged proteins and selenometabolites in human serum has been developed using species-unspecific isotope dilution (SUID)-ICP-QqQ-MS online coupled to 2D/SE-AF-HPLC involving four columns in one run for the analysis of 100 mg of sample.

Using this chromatographic arrangement, the spectral interference produced by bromide and chloride are removed and the total chromatographic runtime is less than 20 min. The analytical approach was validated using a human serum reference material BCR-637 and ClinChek CRM Human Serum Level I and II.

Conventionally, the enriched standard required for isotopic dilution quantification is mixed with the samples or chromatographic flow after HPLC separation using a Y connection. Recently, 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 sense, in this study, we compare the performance of Micromist® and MultiNeb® nebulizers for analytical methodologies optimized in this Application Note for total selenium quantification by IDA-ICP-QqQ-MS or simultaneous quantification of selenoproteins and selenometabolites by (SUID)-ICP-QqQ-MS online coupled to 2D/SE-AF-HPLC.

Performance comparison MultiNeb and MicroMist

2. Experimental

Reagents and solutions

All reagents used in this study were of the highest available purity. The mobile phase solution used in 2D-HPLC-SEC-AF was ammonium acetate (Bioultra grade) purchased from Merck (Darmstadt, Germany), which was prepared daily with ultrapure water (18 MΩcm) obtained from a Milli-Q system (Millipore, Watford, UK) and adjusted to pH 7.4 with ammonia solution, this later prepared by dilution of 20% (v/v) ammonia solution (Suprapur, Merck) with ultrapure water. Standard solutions containing 1000 mg L-1 of Se and 1000 mg L-1 of Br, both stabilized in 5% (v/v) Suprapur nitric acid were purchased from Merck (Darmstadt, Germany).

The human serum certified reference material (CRM) BCR-637 was purchased from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium). Others reference materials were also used for lyophilized human serum (Level I and II) (Recipe Chemicals, Munich, Germany). Enriched 74Se was obtained from Cambridge Isotope Laboratories (Andover, MA, USA) as elemental powder and it was dissolved in the minimum volume of nitric acid (Suprapur grade) and diluted to the appropriate volume with ultrapure water. The concentration of this solution was established by reverse isotope dilution analysis as described elsewhere [3].


A digestor Digiprep® (SCP Science, Canada, model MS-48) was used for the mineralization of serum samples. Selenium trace levels and selenium-linked biomolecules were analyzed with an Agilent Technologies 8800 inductively coupled plasma mass spectrometer (Agilent Technologies, Tokyo, Japan) equipped with a triple quadrupole as analyzer. The 8800 ICP-QQQ was operated in MS/MS mass-shift mode using O2 /H2 at a gas flow of 35% O2 , which translates to 0.35 mL/min and 1 mL/min H2 for a total flow of 1.35 mL/min (combined flow rate). In this study, two nebulizers were evaluated, MultiNeb® (Ingeniatrics Tecnologias S.L., Seville, Spain) and Micromist® (Glass Expansion, Switzerland). Chromatographic separations were performed using a Model 1260 HPLC pumps (Agilent, Wilmington, DE, USA). The complete resolution of selenium species was carried out by using two 5 ml HiTrap® Desalting Columns (GE Healthcare, Uppsala, Sweden), in series connected to two affinity columns, with stationary phases of heparin-sepharose (HEP-HP) and bluesepharose (BLU-HP), both purchased from GE Healthcare, Uppsala, Sweden.

In addition, for speciation analysis based on HPLCICP-MS analytical approach, Ingeniatrics Tecnologias S.L. has released to allow quick, reliable and easy connection of your LC to your ICP, a specific onepiece connector for of MultiNeb® nebulizer (two liquid inlets), the one-piece connectors designed for speciation analysis. One channel of PEEK capillary (30 cm length, 0.75 mm i.d. and 1.6 mm o.d., green PEEK) to connect the exit of the chromatographic column directly to nebulizer and a second channel of PFA tubing (50 cm length, 0.5 mm i.d. and 1.6 mm o.d.) with Part Number: CN2030075 (Ingeniatrics Tecnologias S.L., Sevilla, Spain).

Sample collection and pre-treatment

Human blood samples from the 10 volunteers were obtained by venipuncture of the antecubital region. All samples were collected in BD Vacutainer SST II tubes with gel separator and an advance vacuum system, previously cooled in a refrigerator. The samples were immediately cooled and protected from light for 30 minutes to allow clot retraction to obtain serum after centrifugation (3,500 rpm for 10 minutes). The serum was divided into aliquots in Eppendorf tubes and frozen at -80 °C until analysis.

Total Se determination by isotopic dilution analysis IDA-ICP-QqQ-MS

For the analysis of human serum and certified reference materials, sample amounts of 0.5000 g was weighed directly into the 5.0-mL glass vials and 1.00 mL of nitric acid (65 mass %) and 0.25 mL of hydrogen peroxide (30 mass %) were added. The capped vials were placed in a Digiprep® digestor at 90 °C for 50 minutes. Then, samples were centrifuged, and filtered using PTFE 0.45 µm filter. After that, the solutions were made up to 10.0 g with ultrapure water and the total selenium concentration was analyzed by isotopic dilution analysis (IDA) and ICP-QqQ-MS using the operational conditions summarized in Table 1 and instrument configuration showed in Figure 1. All the analyses were performed using three replicates.

Table I. Operational conditions for IDA-ICP-QqQ-MS
analysis using 8800 ICP-QqQ-MS (Agilent Technologies).

Performance comparison MultiNeb and MicroMist

Figure 2. Schematic representation of both sample introduction systems. A: MultiNeb®-based configuration. B: Micromist®-based configuration for total selenium concentration by IDA.ICP-QqQ-MS.

Conventionally, the 74Se spike required for IDA is mixed with the digested samples using a Y connection when Micromist® nebulizer is employed (Figure 2B). However, 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 (Figure 2A). For online enriched selenium addition using ICP-MS peristaltic pump, orange-white tubings (Part Number: 9910124100, Agilent Technologies) was employed.

Speciation of selenium containing biomolecules by 2D-HPLC-SEC-AFSUID-ICP-QqQ-MS

First, serum samples were filtered through Iso-Disc poly-(vinylidene difluoride) (PVDF) filters (20-mm diameter, 0.2-µm pore size) to avoid column overloading or clogging. After that, speciation of selenium was carried out by 2D/SE-AF-HPLC-SUIDICP-QqQ-MS to separate selenometabolites from high molecular mass selenium containing proteins. In this sense, the fractionation of selenium containing proteins and selenometabolites was carried out by two dimensional chromatographic separations based on double size exclusion prior to double affinity chromatography, using the schematic arrangement shown in the Figure 2.

Performance comparison MultiNeb and MicroMist

Figure 2. Schematic representation of MultiNeb®-based configuration for 2D-HPLC-SEC-AF-SUID-ICP-QqQ-MS using a high-pressure connector for LC-ICP using MultiNeb® Nebulizer (Two Liquid Inlets).

Separation of the analytes was performed in series stacking of two 5 mL HiTrap® Desalting columns in series connected with a dual affinity column arrangement comprising a 1 mL heparin-sepharose column (HEP-HP) and a 1 mL blue-sepharose column (BLU-HP) interconnected by a six-way switching column valve. The HiTrap column is based on size exclusion chromatography, and it is normally used to remove low molecular mass components (MW<1000Da) and high molecular mass molecules, such as DNA, proteins or peptides (MW>5000Da), the combination of two columns increase the resolution of the chromatographic separation. On the other hand, HEP-HP column can retain selectively SeP whereas BLUE-HP column retains both SeP and SeAlb which has been previously described [1-2]. ]Speciation of selenium in human serum by 2D/SEAF-HPLC-SUID-ICP-ORS-MS was carried out using the operating conditions summarized in Tables I and II.

In order to avoid memory effects from previous experiments, the set of polytetrafluoroethylene vessels employed for sample digestion were first cleansed using a microwave procedure, consisting of the addition of 8 g of concentrated HNO3 to each vessel, followed by a microwave treatment at 350 W during 10 min to reach 100 ºC and 10 min at 100 ºC (i.e., 20 min in total). This procedure was performed twice before sample digestion.

Four procedural blanks were also prepared and subjected to the entire analytical methodology together with the fish homogenate replicates in order to evaluate the contribution of the blank contamination. Six replicates were prepared for the analysis.

Table II. Chromatographic conditions of 2D/SE-AF-HPLC using 1260-HPLC (Agilent Technologies).

Chromatographic performance was checked regularly by measuring control standards to ensure enough separation between species and method sensitivity after each five analysis. All samples and CRM were analyzed by duplicated.

Ammonium acetate was used as mobile phase with a gradient from 0.05 mol L-1 (pH 7.4) to 1.5 mol L-1 (pH 7.4), at a flow rate of 1.3 ml min-1 with an injection volume of 100 µL. Serum samples aliquots were injected in position A (Figure 2A) with mobile phase A passing through the system. Then, selenium containing proteins (eGPx, SeAlb and SeP) are separated by SEC (HiTrap columns) from low molecular weight selenium species and bromide and chloride interferences. Then, SeP is retained in the HEP-HP column and SeAlb in the BLUE-HP one. eGPx, selenometabolites and Br+Cl interferences were not retained in affinity columns and elute resolved in three peaks. After the retention step, mobile phase B was pumped through the system in position B (Figure 2B) for the elution of SeP. Finally, the valve was switched again to position A for the elution of SeAlb. After elution, mobile phase A was again passed through the system for equilibration during 10 minutes prior to the next injection.

On the other hand, when Micromist® nebulizer is employed, the HPLC-SUID-ICP-QqQ-MS online coupling was performed by connecting the outlet of the chromatographic column to the Y connector direct to Micromist® nebulizer inlet (Glass Expansion, Switzerland) of the ICP-MS by means of a 30 cm PEEK tubing (0.75 mm i.d. and 1.6 mm o.d., green PEEK). Post column isotope dilution analysis was performed by the introduction of 74Se via the Y connector (Figure 3). Quality control of the HPLCSUID-ICP-ORS-MS system to overcome problems related to contamination, loss and stability of species has been performed the procedure described elsewhere [4]. Separation of the analytes was performed using the same chromatographic conditions employed using MultiNeb®-based configuration previously described.

Performance comparison MultiNeb and MicroMist

Figure 3. Schematic representation of Micromist®-based configuration for 2D-HPLC-SEC-AF-SUID-ICP-QqQ-MS using a Y connector for LC-ICP (One Liquid Inlets).

The quantification of selenium containing proteins and selenometabolites in the different chromatographic peaks was carried out by postcolumn specie-unspecific isotopic dilution analysis as described by C. Sariego-Muñíz et al., 2001 [5]. Briefly, the intensity of different selenium isotopes and polyatomic interferences were converted to mass flow chromatogram for the quantification of the selenium species in plasma and serum samples. Mathematical treatments were applied to correct BrH+ and SeH+ polyatomic interferences. Mass bias corrections were applied using the 78Se/74Se and 80Se/74Se isotope ratios calculated (exponential mode) as previously described by J. Ruiz-Encinar et al. [6]. Finally, an online dilution equation was applied to each point of the chromatogram and the amount of selenium in each chromatographic peak calculated using Origin 8.5.1. software (Microcal Software Inc., Northampton, MA, USA).

During the chromatographic separation, the isotopes 74Se, 77Se, 78Se, 80Se, 82Se, 79Br and 83Kr (82Se1H+ ) were simultaneously online monitored by ICP-QqQMS, detected as SeO+ in O2+H2 mode to ensure the absence of isobaric interference (Figure 4).

Figure 4. Illustration of O2 mass shift method of ICPQqQ-MS (Agilent Technologies).

The use of two small SEC chromatographic columns arranged in series (Hiptrap® Desalting Column) allows a good resolution in the speciation of eGPx and selenometabolites, with retention times about 2 and 4 min, respectively. In addition, efficient separations of SeP (11 min) and SeAlb (16 min) in undiluted human serum are obtained using an online dual chromatographic affinity column arrangement that exploits the selenoproteins selectivity towards Heparin-sepharose and Blue-sepharose stationary phases (Figure 5).

Figure 5. Mass flow chromatogram 78Se/74Se isotope ratios in human serum using 2D/SE-AF-HPLC-SUID-ICPQqQ-MS.

3. Results and discussion

For selenium containing biomolecules quantification, numerous speciation methods based on liquid chromatographic separations have been developed, being SEC the most widely used due to the high reproducible results and remarkable tolerance to complex matrix such as biological fluids (e.g. serum/plasma). Nevertheless, low resolution for selenium containing proteins is obtained, and alternatively, a number of methods introduce a heparine-sepharose column prior to SEC chromatographic separation in order to improve the resolution by retention of SeP, but the time of analysis increases to 60 minutes [7]. On the other hand, good recoveries were obtained using anionic exchange chromatography, but the chromatographic resolution was not adequate [8]. In this way, the present study, which combines two SEC columns with two AF columns, allows efficient separations of eGPx, SeAlb, SeP and low molecular mass selenium species in less than 20 minutes avoiding chlorine and bromide polyatomic interferences (Figure 5). 

The proposed speciation method has been validated using some CRM of human serum (BCR-637 and Clinchek Human Serum Level I and II). These materials were additionally spiked with 25 µg kg-1 of inorganic selenium (sodium selenate) to evaluate the recovery and precision of method. Interconversion among selenium species was not observed under detailed experimental condition.

For evaluate the signal stability along the sequence of analysis, a monitoring standard solution (BCR-637) spiked with 25 µg kg-1 of inorganic selenium (sodium selenate) was prepared. This solution was analyzed once every five samples, in order to evaluate the stability of the signal. The recoveries must fall within the limits of 97-106 %. Additionally, the stability of the retention time was studied. The stability of retention time was achieved in the range of 0.2–2.2% (short term, n = 5) and 0.5–4.5% (long term, n=20) for all selenium containing biomolecules.

MultiNeb® nebulizer use Flow Blurring nebulization technology instead of the traditional Venturi effect, as Micromist® nebulizer. 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.

In addition, 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 III. Additionally, quantification of selenium containing biomolecules has been performed in 10 human serum using the proposed speciation method 2D/SE-AF-HPLC-SUID-ICP-QqQMS. Finally, precision values were evaluated using different certified reference materials (CRM) following the analytical procedure previously described.

The results obtained are shown in Table III. In summary, as observed in Table III, the new MultiNeb® multiple nebulizer exhibits higher precision, sensitivity, signal stability and reproducibility in selenium determination using IDAICP-QqQ-MS and selenium speciation quantification based on 2D-HPLC-SEC-AF-SUID-ICP-QqQ-MS.

Table III. Experimental and certified mean values for total selenium and selenium species concentrations in the different certified reference materials and human serum samples, as well as the RSD obtained for 2 replicates using 2D-HPLCSUID-ICP-QqQ-MS and 3 replicates for IDA-ICP-QqQ-MS using MultiNeb® and Micromist® nebulizers.

4. Conclusions

In this study, it has been demonstrated that the new MultiNeb® multiple nebulizer presents higher precision, sensitivity, signal stability and reproducibility in total selenium determination using IDA-ICP-QqQ-MS and selenium containing biomolecules quantification based on two dimensional HPLC-SUID-ICP-QqQ-MS.

On the other hand, the high-pressure connector designed by Ingeniatrics Tecnologias S.L. has several advantages, such as resistance to blockage, fast washout, minimized dead volume, and peak broadening, among others. Additionally, this connector is easy to use and allows for a quick, reliable, and easy connection of your HPLC to your ICP instrument.

5. References

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2. MA García-Sevillano, T García-Barrera, J L GómezAriza, J. Chromatogr. A. 1318 (2013) 29.

3. L Hinojosa-Reyes, JM Marchante-Gayón, JI GarcíaAlonso, A Sanz-Medel, J. Anal. At. Spectrom. 18 (2003) 1210.

4. M González-Fernández, MA García-Sevillano, R JaraBiedma, T García-Barrera, A Vioque, J López-Barea, C Pueyo, JL Gómez-Ariza, J. Anal. At. Spectrom. 26 (2011) 141.

5. C Sariego-Muñíz, JM Marchante-Gayón, JI GarcíaAlonso, A Sanz Medel, J. Anal. At. Spectrom. 16 (2001) 587.

6. F Vanhaecke, G Wannemacker, L Moens, R Dams, C Latcoczy, T Prohaska, G Stingerer, J. Anal. At. Spectrom. 13 (1998) 567.

7. H Koyama, K Omura, A Ejima, Y Kasanuma, C Watanabe, H Satoh, Anal. Biochem. 267 (1999) 84.

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