MultiNeb®: Arsenic speciation in foods and human biological fluids using HPLC-ICP-MS by online internal standard correction technique

1. Introduction

Arsenic has been recognized as a global toxic element that affects human health and can be the promoter of some types of cancers such us skin, lung, urinary bladder and liver cancer [1]. Humans can be exposed to arsenic by contaminated water, soil, atmosphere and food, especially seafood and cereals, such as rice [2]. Once ingested most of the arsenic is excreted from the body, but a certain fraction of its accumulates in the body giving rise to several health effects including cardiovascular effects, cancer and hypertension.

As toxicity is strongly related to the chemical species in which it is present. In mammals, inorganic forms of arsenic (arsenate (AsV) and arsenite (AsIII)) are more toxic, while methylated forms, MMAV (monomethylarsonate) and DMAV(dimethylarsinate), are considered only moderately toxic [3-4]. In addition, other arsenic species, like trimethyl-arsine oxide (TMAO) and tetra-methyl-arsonium (TETRA) are also considered moderately toxic, whereas arsenobetaine (AsB), arsenocholine(AsC) and other arsenosugars (AsS) show no toxicity [5]. In addition, iAsIII is more toxic than iAsVmost probably due to enhanced cellular uptake and accumulation of the former (LD50for mice are 4.5 mg kg-1 and 14-18 mg kg-1for the arsenite and arsenate, respectively) [6].

Therefore, the development of accurate and precise arsenic speciation procedures, for biological and environmental materials, is a current trend in analytical chemistry. In this sense, the hyphenation of highperformance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICPMS) is a very versatile and powerful tool for arsenic speciation analysis. However, while ICP-MS as a standalone instrument allows for high sample throughput for multi-element determinations, its coupling with a chromatographic separation system is reducing the sample throughput significantly. Also, the perturbations in the pressure pump, nebulization process and the mobile phase of HPLC might create some plasma instability, especially when changing its composition during gradient elution. As a result, the detection response and the baseline signals often fluctuate during analysis, limiting precision and accuracy of measurements. 

An approach to correct drift and measurement fluctuations is the use of an internal standard, which is usually used in ICP-MS. The selection of the internal standard element is critical (IS is not contained in significant amounts in the samples and that the chosen element behave similarly to the analyte with respect to plasma fluctuations. When large number of samples must be analyzed, the addition of the internal standards to all the samples is a timeconsuming step that also may cause some experimental errors. For the stand-alone ICP-MS the solution to this problem is the on-line addition of the internal standard after chromatographic separation.

In this study, an online internal standard correction technique for high-performance liquid chromatographyinductively coupled plasma mass spectrometry (HPLCICP-MS) was optimized using an HPLC-ICP-MS to improve the analytical precision. For this purpose, we evaluate in this study the performance of MultiNeb® inert, robustness and durability nebulizer and 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.

MultiNeb arsenic speciation foods

2. Experimental

Reagents and solutions

All reagents used for sample preparation were of the highest available purity.

The chemical standards used were AB (95%, Fluka, Steinheim, Germany), AsIII and AsV(99%, Merck, Darmstadt, Germany), DMA, MA (99%, Supelco, Bellefonte, USA). All the standards were dissolved in doubly deionized water (18 MΩ cm) obtained with a Milli-Q Gradient system (Millipore, Watford, UK) and stored at 4 ºC in the dark. Working solutions were prepared daily by appropriate dilution of the stock solutions at 1000 mg L-1. For total element determination and arsenic speciation analysis, nitric acid (65 mass %), hydrogen peroxide (30 mass %) and trichloroacetic acid were of the highest availablepurity (Merck, Darmstadt, Germany) were used for sample preparation. All standard solutions were prepared daily. The arsenic concentrations of the AsB, DMA and MMA standards were verified using ICP-MS analysis. The total arsenic concentrations of 1 µg g-1MMA and DMA standards were determined by online internal standard calibration. These concentrations were used to recalculate the stock standard concentrations and the new values were then applied to the future calculations. Certified Reference Materials of dogfish muscle (DORM-2) and lobster hepatopancreas (TORT-3) (National Research Council Canada) and rice (ERM-BC211, JRC Institute for Reference Materials and Measurements) were used for quality control assays. Reference materials were also used for lyophilized urine (Clinchek of urine control, Level II) and serum (Level II) (Recipe Chemicals, Munich, Germany).

Ammonium carbonate (NH4)2CO3(Fluka), ammonium hydroxide (Sigma-Aldrich), and ethylenediaminetetraacetic acid disodium salt (Na2EDTA) were used in the mobile phases for the chromatographic separation. Methanol and acetonitrile were purchased from Aldrich (Steinheim, Germany).

The aqueous calibration standards for total arsenic determination by ICP-MS were prepared by appropriate dilution of a mono-elemental stock solution of 1000 mg L-1of As (ICP CetriPUR, Merck, Darmstadt, Germany) in deionized water (18 MΩ cm resistivity). All aqueous solutions are acidified by adding up to 5% nitric acid. In addition, Yb, Rh, Re, and Te were investigated as internal standards, since they are unlikely to be contained in food and human biological fluids samples. A solution containing internal standard is prepared by appropriate dilution of a 1000 mg L−1 of mono-elemental stock solutions of each internal standard element investigated in this work (high-purity mono-element standard solutions).


Anion-exchange chromatography (AEC) analysis was carried on an Agilent 1260 HPLC system comprising a quaternary pump, autosampler and vacuum degasser was coupled to an Agilent 7900 CP-MS. Arsenic species were separated on a Hamilton PRP-X100 anion-exchange column (250 × 2.1 mm × 10 µm particle size) with a preventive guard column (30 × 2.1 mm × 10 µm particle size). Samples and standard solutions were injected via a 100 µL sample loop. Temperature of the column was kept at 30°C during chromatographic separation. Arsenic compounds were separated by employing a gradient of mixture of 5 mM (NH4 )2CO3 , pH 9.0 (adjusted by using NH4OH), 0.05% Na2EDTA (Chanel A) and 50 mM (NH4)2CO3 , pH 9.0 (adjusted by using NH4OH), 0.05% Na2EDTA and 5.0 % of MeOH (Chanel B). The program of the mobile phase was set up as follows: the concentration of (NH4 )2CO3 was 5 mM for 2 min followed by a linear increase to 50 mM in 6 min. After keeping at 50 mM for 4 min, the concentration of (NH4 )2CO3 was then decreased to 5 mM in 1 min and kept for 5 min for reequilibration of the column for the next injection. The flow rate of mobile phase was kept constant at 700 µL·min−1 during chromatographic separation. Total time for separating of arsenic species was 10 min.

The Agilent 7900 ICP-MS (Agilent Technologies) with the Octopole Reaction System (ORS4) collision/reaction cell (CRC) provides sensitive and specific analysis of As in the presence of multiple interferences. In this sense, the formation of polyatomic ions based on ArCl and CaCl may cause spectral interferences on the sole isotope of As at m/z 75. However, operating the 7900 ICP-MS ORS4 in helium mode effectively removes these matrixbased polyatomic interferences on As.

The MultiNeb® nebulizer used in this study consists of two independent liquid inlets and a common gas inlet in a single nebulizer body of polytetrafluoroethylene (Figure 1) and specifications are shown in Table I).

MultiNeb arsenic speciation foods

Figure 1. MultiNeb® nebulizer (Ingeniatrics Tecnologías S.L.)

Table I. MultiNeb® nebulizer specifications (Ingeniatrics Tecnologias S.L.)

In addition, for speciation analysis based on HPLCICP-MS analytical approach, Ingeniatrics Tecnologías S.L. has released to allow quick, reliable and easy connection of your LC to your ICP, a specific one-piece connector for OneNeb®or MassNeb®. (one liquid inlet) with a 50 mm PEEK capillary to connect the exit of the chromatographic column directly to nebulizer (for high and low pressure, red and green PEEK capillary, respectively), replacing the conventional HPLC-ICP-MS connection kit employing for this purpose (Figure 2A). In addition, in case of MultiNeb®nebulizer (two liquid inlets), the one-piece connectors designed for speciation analysis contains a 50 mm PEEK capillary to connect the exit of the chromatographic column directly to nebulizer and a second 50 mm PFA tubing (0.5 mm i.d.) (Figure 2B). In this study, speciation analysis high pressure connector for LC-ICP using MultiNeb®Nebulizer (Two Liquid Inlets) has been used (Figure 2B left). Speciation Analysis High Pressure Conectors for LC-ICP designed by Ingeniatrics Tecnologías S.L. present some advantages, such as resists blockage, fast washout, minimize dead volume and peak broadening, principally. In addition, these connectors are simple to use, to allow quick, reliable and easy connection of your HPLC to your ICP instrument.

MultiNeb arsenic speciation foods

Figure 2. A) Speciation Analysis Connector for HPLC-ICP using OneNeb® or MassNeb® Nebulizers (One Liquid Inlet) and B) Speciation Analysis Connector for LC-ICP using MultiNeb® Nebulizer (Two Liquid Inlets).

The new MultiNeb® nebulizer is the only two-channel analytical nebulizer on the market and is built on the right dimensions to allow easy connection to any commercial spray chamber conventionally used in ICP-based. Allows the simultaneous nebulization of both solutions, opening the door to new analytical methods. Recently, it has been demonstrated that this fact makes MultiNeb® nebulizer more appropriate for several applications, such as hydride generation, isotopic dilution analysis, standard addition quantification, internal standard calibration for total multielement determination using ICP-OES and ICP-MS detectors, and others. In this study, MultiNeb® nebulizer is proposed as an appropriate nebulizer in speciation analysis based on the use of HPLC-ICP-MS instrument configuration by online internal standard correction technique using the OnePiece High Pressure for Speciation Analysis by LCICP (Two Liquid Inlets (Part Number: CN.200300.005, Ingeniatrics Tecnologías S.L.).

Additionally, the choice of an appropriate calibration method is critical for the compensation of physical and/or spectral interferences for obtaining accurate results.

Conventionally, for total element determination by internal calibration, the internal standard is mixed with the calibration standards and samples using a Y connection. 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 (Figure 3).

MultiNeb arsenic speciation foods

Figure 3. Schematic representation of MultiNeb®-based configuration for total element determination by internal standard calibration technique by ICP-MS.

MultiNeb arsenic speciation foods

Figure 4. Schematic representation of MultiNeb®-based configuration for Speciation Analysis using a highpressure connector for LC-ICP using MultiNeb® Nebulizer (Two Liquid Inlets).

In speciation analysis based on the use of HPLC-ICPMS, signal stability and plasma drift, also the perturbations in the pressure pump, nebulization process, nebulizer blockage and the mobile phase of HPLC might create some plasma perturbations, especially when changing its composition during gradient elution, etc. As a result, the detection response and the baseline signals often fluctuate during analysis, limiting precision and accuracy of quantifications. In this sense, with order to reduce the effects previously mentioned, in this work, an analytical methodology has been proposed for arsenic speciation analysis using HPLC-ICP-MS by online internal standard correction technique based on instrumental schematic representation of MultiNeb® -based configuration showed in Figure 4. For online internal standard addition using ICP-MS peristaltic pump, orange-white tubings (Part Number: 9910124100, Agilent Technologies) were employed in this study. The precision of the online internal standard technique primarily depends on the precision of the introduction of internal standard flow. For this purpose, 20 rpm was selected for peristaltic pump rate. In addition, Yb, Rh, Re, and Te were investigated as internal standards, since they are unlikely to be contained in food and human biological fluids samples. Re is supplied in nitric acid solution and the stable isotopes are 185Re and 187Re with an abundance ratio of 37 : 63. Therefore, Re as mainly used as the internal standard in this study. The fluctuations of As and Re signals obtained by HPLC-ICP-MS were monitored. The results obtained are shown relative standard deviation of the signal intensity ratios 75As/185Re was suppressed.

Operating conditions for total element determination using ICP-MS by internal standard calibration and speciation analysis by anion-exchange chromatography (AEC) combined with inductively coupled plasma mass spectrometry (ICP-MS) for arsenic species quantification optimized are indicated in Table II.

Table II. Operational conditions for 7900 ICP-MS and 1260 HPLC optimized for arsenic speciation by online internal calibration technique (Agilent Technologies).

Sample preparation

For determination of total As concentration 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. 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 pore-size). The total content of arsenic was measured by ICP-MS using as internal standard rhodium (Re) to 5 µg L-1. A blank with the reagents used for total arsenic determination was run simultaneously to the sample preparation.

On the other hand, for speciation analysis, he sample extraction and clean-up procedures constitute a crucial step when biota samples are considered, due to possible analyte losses, changes of the species or incomplete extraction of the arsenic compounds, which may lead to poor or erroneous results.

For arsenic species extraction from the rice samples (1.0 g) was weighed into the 50.0-mL glass vials and 10 mL of 0.28 mol/L HNO3 was added. On the other hand, for lobster and dogfish samples (0.5 g) were weighted into the 50.0-mL glass vials and 10.0 mL of MeOH and H2O mixed solution (8:2, v/v) was added. The capped vials were placed in a Digiprep® digestor at 90 °C for 50 minutes. Then, samples were centrifuged, and filtered. Finally, for rice samples, 0.5 mL portions of rice extract were pipetted in to a 2 mL plastic HPLC vials and was diluted with 1.5 mL of mobile phase (Channel A). For dogfish and lobster samples, 1.0 mL portions were pipetted in to a 2 mL plastic HPLC vials and was diluted with 1.0 mL of mobile phase (Channel A).

For serum samples, protein in the serum sample was precipitated as follows: 400 µL of serum sample was accurately weighed, and 500 µL of 25% of trichloroacetic acid was added and then treated with 50 µL of acetonitrile and 50 µL deionized water, followed by being vortexed for 120 s. The mixture was then centrifuged at 10000×g for 10 min at 4°C. Clear aliquots of the supernatant were injected into HPLC-ICP-MS system.

For urine samples, a tenfold dilution of the samples was carried out with a mixture of deionized water and methanol (9/1, v/v). This solution was subjected to HPLC-ICP-MS analysis. For speciation analysis each sample was prepared in duplicate, and all extracts were filtered prior to analysis by HPLC-ICPMS. All the filters were cleaned with 5 mL of the extracting solution before the use to avoid the contamination of the samples.

3. Results and Discussion

Arsenic speciation analysis

For arsenic speciation analysis, many studies used HPLC-ICP-MS as an effective method. Because of ionic compounds, either anion-exchange chromatography or ion pair chromatography has been employed for speciation of arsenic compounds. However, most of the mobile phases including phosphate buffer for anion-exchange chromatography or carbon rich mobile phase for reversed phase chromatography were used. As a major drawback, carbon build up and ion suppression commonly affected mobile phases in case of ion pair chromatography or influenced the physical properties of the sample introduction devices in case of anion-exchange chromatography employing high concentration of buffer, e.g., phosphate buffer. In this study, we the mobile phase including ammonium carbonate, methanol, and Na2EDTA was adjusted to pH 9 and used for separation of five arsenic species.

In recent years, Na2EDTA is added to mobile phase to obtain better peak shape and resolution have previously been reported in several studies of arsenic speciation and has demonstrated that it prevented the loss of the arsenic compounds during the chromatographic separation, especially iAsIII. For this purpose, in this study, 0.05% EDTA (m/v) was added to the mobile phase. In addition, the organic modifiers in mobile phase such as methanol and ethanol have been reported as improvement reagents for better chromatographic resolution and increased sensitivity when ICP-MS is employed as a detector. In this study a 5% of MeOH was selected.

Interconversion among arsenic species was not observed under detailed experimental condition. In addition, all five arsenic species were speciated under operational and experimental conditions optimized, previously described (Figure 5).

Figure 5. Chromatogram of five arsenic species speciatedusing HPLC-ICP-MS (10 ng mL−1 according to As for eachform).

The isobaric mass of polyatomic interferences, which can come from the argon-based plasma such as 40Ar35Cl+ is also a main drawback of quadrupole mass analyzer. To overcome the polyatomic interference in arsenic measurement, either dynamic reaction/collision cell or high-resolution ICP-MS was employed. In this study, for the assessment of mass interference on the arsenic measurement, 100 µg·mL−1 of chloride was prepared in deionized water and injected on the HPLC-ICP-MS at the above condition. The chloride ion was eluted at 4.31 min, before to MMA with depreciable intensity. In addition, the peak of chloride ion was separated far from all arsenic species peaks using optimized chromatographic conditions. It should be noted that high chloride matrices such as human urine sample is important for arsenic speciation. The spiked experiments of chloride in real sample matrices (urine and serum) were also carried out, and the result showed that there was no statistically significant effect of chloride ion on the quantification of arsenic species in such matrices.

Online internal standard calibration technique

In speciation analysis based on the use of HPLC-ICPMS, signal stability and plasma drift, also perturbations in the pressure HPLC pump, nebulization pressure alterations, nebulizer blockage, progressive clogging of the ICP-MS interface from total dissolved solids (TDS) contained in samples and mobile phase, changes in mobile phase composition causing perturbations on plasma signal response, especially when changing its composition during gradient elution and as result alterations on nebulization process efficiency and/or perturbations on the plasma ionization power related with modification in matrix composition and therefore in its chemical-physical properties (density, viscosity, solubility, and others), etc. In this sense, it has been demonstrated that MeOH increases the signal response when ICP-MS is used as a detector.

In this study, with order to reduce the effects previously mentioned, the fluctuations of 75As and 185Re signals were investigated in order to confirm the efficiency of online internal standard quantification using 185Re as internal standard to reduce the deviations in the measurements. For this purpose, triplicated solutions of 5 µg L-1 of each arsenic species with increased proportion of some components of mobile phase, mainly (NH4 )2CO3 and MeOH, from 0 – 50 mM and 0.0 – 5.0 %, respectively. In contrast, the content of 0.05% Na2EDTA was the same for all solutions.

The final solutions were analyzed by ICP-MS. The relative standard deviations of 75As and 185Re measurements in triplicate were 2 – 4 %, the relative standard deviation of the signal intensity ratios 75As/185Re was suppressed to 1 – 2 %.

Additionally, a progressive increment in the baseline signal for both isotopes analyzed were obtained in all analysis, more pronounced in blank samples. A similar response was observed in the HPLC pressure pump along the elution time. This fact could be related with a gradient elution used for arsenic speciation analysis in this study. This observation was smoothing when the signal intensity ratios 75As/185Re was represented. The standard deviation of the baseline signal when 75As/185Re ratio was considered was decreased.

Signal stability and sensitivity

For evaluate the signal stability along the sequence of analysis, a monitoring standard solution containing 5 µg g-1 of this element 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 96-104 %. Additionally, the stability of the retention time was studied. The stability of retention time was achieved in the range of 0.1–2.0% (short term, n = 5) and 0.4–6.0% (long term, n=20) for all AsB, As(III), MMA, DMA, and As(V).

MultiNeb®nebulizer use Flow Blurring nebulization technology instead of the traditional Venturi effect. 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 this sense, different flow rates for nebulization flow and peristaltic pump speed were checked, obtaining the best results for 0.6 L min -1 and 20 rpm, respectively. 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.

The results obtained shown a very good relation between analytical signal 75As/185Re ratio and concentration of arsenic species (R2> 0.9995 in all cases). The regression coefficient obtained were increased when Re is used as internal standard.

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 III Chromatograms obtained are shown in Figure 6.

To validate the method performance in real samples, a spike recovery test was performed using the mixed As species standard solution. The recoveries of AsB, As(III), MMA, DMA, and As(V) obtained in all cases were 91-107 %.

Table II. Experimental and certified mean values for total arsenic and arsenic species concentrations in the different foods and human biological fluids analyzed, as well as the RSD obtained for 2 replicates using HPLC-ICP-MS and 5 replicates for ICP-MS determination using MultiNeb® nebulizer by online internal standard calibration technique (Grey: certified values; Green: Experimental results).

MultiNeb arsenic speciation foods

Figure 5. Chromatograms obtained by HPLC-ICP-MS with online internal standard calibration technique using MultiNeb® for the different foods and human biological fluids analyzed in this study.

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 arsenic determination using ICP-MS and arsenic speciation quantification based on HPLC-ICP-MS coupling by online internal standard calibration technique. Additionally, the detection of the ICP-MS is element specific, and therefore, when using isocratic HPLC conditions, it is sufficient to use a single elemental standard as a calibrant for all species. However, it is not possible when a gradient elution is required as mobile phase. For this purpose, the effect of Re as the internal standard element has been validated and confirmed for all species and mobile phase composition.

In summary, the innovative method optimized in this work for arsenic speciation could be extrapolated to online quantification using Isotopic Dilution Analysis (IDA).

5. References

1. A.H. Smith, M. Goycolea, R. Haque, M.L. Biggs, Am. J. Epidemiol. 147 (1998) 660.

2. K. Francesconi, Pure Appl. Chem. 82 (2010) 373.

3. A. E. Geisinger, W. Goessler, K. Francesconi, Marine Environ. Res. 53 (2002) 37.

4. D. Fattorini, F. Regoli, F. Environ. Toxicol. Chem. 23 (2002) 1881.

5. D. Fattorini, A. Notti, A., F. Regoli, Chem. Ecol., 22 (2006) 405.

6. Yamauchi H, Fowler B.A., (1994) Nriagu JO (ed) Arsenic in the environment, part II: Human health and ecosystems. JohnWiley&Sons, Ann Arbor, p35.

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