Analytical and Bioanalytical Chemistry

, Volume 377, Issue 1, pp 132–139 | Cite as

Determination of sulfur and selected trace elements in metallothionein-like proteins using capillary electrophoresis hyphenated to inductively coupled plasma mass spectrometry with an octopole reaction cell

Original Paper

Abstract

The determination of sulfur in biologically relevant samples such as metalloproteins is described. The analytical methodology used is based on robust on-line coupling between capillary electrophoresis (CE) and octopole reaction cell inductively-coupled plasma mass spectrometry (ORC–ICP–MS). Polyatomic ions that form in the plasma and interfere with the determination of S at mass 32 are minimised by addition of xenon to the collision cell. The method has been applied to the separation and simultaneous element-specific detection of sulfur, cadmium, copper, and zinc in commercially available metallothionein preparations (MT) and metallothionein-like proteins (MLP) extracted from liver samples of bream (Abramis brama L.) caught in the river Elbe, Germany. Instrumental detection limits have been calculated according to the German standard procedure DIN 32645 for the determination of sulfur and some simultaneously measured trace elements in aqueous solution. For sulfur detection limits down to 1.3 μg L−1 (34S) and 3.2 μg L−1 (32S) were derived. For the other trace elements determined simultaneously detection limits ranging from 300 ng L−1 (58Ni) to 500 ng L−1 (66Zn, 55Mn) were achieved. For quantification of sulfur and cadmium in a commercially available MT preparation under hyphenated conditions the use of external calibration is suggested. Finally, the need for proper sample-preparation technique will be discussed.

Keywords

Inductively coupled plasma mass spectrometry Capillary electrophoresis Hyphenation Collision cell Metallothionein Speciation Biomolecules 

Introduction

Since their discovery in 1957, metal-binding proteins such as metallothioneins (MT) have been at the focus of research in biology and medicine because of their various functions in connection with the transport, storage, and detoxification of both essential and toxic trace elements in different organisms. One characteristic feature of all MT is the occurrence of Cys-xAA-Cys tri-peptide sequences, where xAA represents an amino acid residue other than cysteine. As a result, mammalian Class 1 MT contains 20 cysteine residues which cause the high affinity of this protein group for up to 12 mono (Cu+, Ag+) and seven divalent (Hg2+, Cd2+, Zn2+) metal ions. Due to genetic polymorphism, MT may be found as different isoforms which differ slightly in their amino acid sequences and therefore have different chromatographic and electrophoretic properties [1, 2, 3, 4].

The most challenging analytical task, besides extraction and separation of the different MT isoforms, is the development and application of element- and isoform-specific detection and quantification techniques due to the different biological functions suggested for different isoforms [5]. Solving these problems requires efficient chromatographic or electrophoretic separation techniques in combination with sensitive element-specific detection methods. One unique property of all MT isoforms, besides their high metal binding capacity, is the high sulfur content found in these protein groups. Sulfur is the characteristic element found in the amino acids cysteine and methionine which occur 20 times and once, respectively, in a normal MT isoform consisting of 61 amino acids. Therefore the sulfur stoichiometry in different MT is constant whereas the composition of the bound trace elements may be different depending on the isoform, the state of the organism investigated, and its role in the whole metabolism. The known sulfur content offers the possibility to characterise the stoichiometric composition of MT isoforms and to quantify them.

Techniques such as flame photometry [6], total X-ray fluorescence spectrometry (TXRF) [7] and inductively coupled plasma (ICP) based detection techniques such as inductively coupled plasma–optical emission spectroscopy (ICP–OES) [8, 9] or inductively coupled plasma mass spectroscopy (ICP–MS) have been used for the determination of sulfur in different kinds of sample.

ICP–MS is one of the most sensitive methods for the simultaneous determination of trace elements in different matrices. However, one of the main drawbacks of ICP–MS is the formation of polyatomic ions in the 7000 K argon plasma generally used. Due to interferences caused by polyatomic ions at the mass of 32S (16O16O+, 31.98983 amu; 14N18O+, 32.00223 amu; 15N16O1H+, 32.00285 amu), high resolution ICP–MS is required in order to resolve the interferences from the elemental peak in the mass spectrum. A medium resolution of about mm=4000 is sufficient for resolving the mass of 32S (at exactly 31.97207 amu) from the interferences. Prohaska et al. used ICP–SFMS combined with a membrane desolvation system for precise sulfur isotope ratio measurements in environmental samples [10]. In combination with HPLC, ICP–SFMS was used for the determination of sulfur/phosphorus ratios in protein samples or for the sulfur-specific detection of impurities in sulfur-containing drugs [11, 12].

Quadrupole mass analyser-based ICP–MS systems have also been used for sulfur determination, either under normal or hyphenated conditions. Therefore the ICP–MS was tuned to generate high oxide ratios. Under more or less reproducible conditions, sulfur was measured as 32S16O+ using the corresponding m/z value of 48 for which interference is less [13, 14].

A new approach to the determination of non-metals with ICP mass spectrometry involves the use of collision or reaction cells in combination with less expensive quadrupole mass spectrometers in order to reduce interferences via chemical reactions or interactions with collision gases.

In general, two different approaches to the chemical resolution of interferences can be applied in the determination of 32S. The first approach is based on the chemical reaction of the analyte masses with reactive gases in order to create product ions which interfere less than the original masses. The use of gases such as N2O, CO2 or O2 for oxidation, or CH4 and C2H4 for methylation has been described. For the analysis of 32S, oxygen was successfully used to convert sulfur ions via an exothermal reaction into the masses of 32S16O+ (m/z 48) which interfere less [15]. The second approach is based on the direct measurement of the most abundant analyte mass of 32S after removal of the interfering ions by using gases such as H2, He or Xe.

Lobinski et al. and, recently, Prange and Schaumlöffel reviewed the current state of different techniques for the analysis and quantification of MTs [16, 17].

A number of workers have demonstrated the use of the isotope dilution technique for the quantification and characterisation of different metallothionein isoforms [18, 19] using capillary zone electrophoresis hyphenated with ICP–SFMS.

This paper will focus on the optimisation of an octopole reaction cell ICP–MS hyphenated with capillary electrophoresis for application to the separation and simultaneous element-specific determination of sulfur, copper, zinc, and cadmium in commercial MT preparations and in metallothionein-like proteins (MLP) extracted from bream liver samples, using Xe as a collision gas for minimisation of the interferences at the mass of 32S.

Experimental

Chemicals and materials

Ultra-pure water (18 MOhm cm−1) was obtained from an Elix 3/Milli-Q Element system (Millipore, Milford, MA, USA). Nitric acid (Suprapur, 65%) and hydrochloric acid (Suprapur, 30%) were both obtained from Merck (Darmstadt, Germany). Both were cleaned by sub-boiling distillation in a quartz apparatus (Kürner, Rosenheim, Germany) and diluted with ultra-pure water to the desired concentration level. Ammonia solutions were prepared by dilution of 25% ammonia solution (Suprapur, Merck) with ultra-pure water. Potassium hydroxide solutions (1 and 0.1 mol L−1 KOH) were prepared by dissolving KOH·H2O (Suprapur, Merck) in ultra-pure water. Tris buffer solution was prepared by dissolving Tris(hydroxymethyl)aminomethane (p.a., Merck) in ultra-pure water and adjusting the solution with nitric acid to the desired pH. Hydrogen (H2, 5.0, 99.999%), helium (He, 5.0, 99.999%) and Xenon (Xe, 4.8, 99.998%) were purchased from Messer Griesheim (Krefeld, Germany). Rabbit liver metallothionein (MT, Lot 19H7812) and rabbit liver MT-1 (Sigma MT-1, Lot 127H7810) were purchased from Sigma (Deisenhofen, Germany). A single-element standard of S (1000 mg L−1) and a multi-element standard (Merck IV) were both Certipur for ICP–MS and purchased from Merck. Cadmium nitrate tetrahydrate (p.a.) was also purchased from Merck. Thiourea was purchased from Sigma (Sigma, Deisenhofen, Germany). Chelex 100 ion-exchange resin (Sigma) was used for buffer purification. Ultra filtration units (Viva Spin 100 kDa MWCO PES) were purchased from Satorius Viva Science (Göttingen, Germany). Units with a MWCO of 3 kDa were purchased from Microcon (Microcon Millipore, Bedford, MA, USA).

Instrumental set-up

The instrumental set-up for all CE–ICP–MS experiments consists of an Agilent 3D capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) and an Agilent 7500c quadrupole ICP–MS with a shield-torch system and an octopole reaction system (Agilent Technologies, Tokyo, Japan). Both instruments were coupled via an interface based on a modified micro-concentric nebulizer (CEI 100, CETAC Technologies, Omaha, Nebraska, USA). Details of the interface construction can be found in previous publications [20, 21]. The CE–ICP–MS interface was adjusted to give a continuous flow rate of about 10 μL min−1 in order to minimise the intake into the plasma. A 10 mmol L−1 ammonia solution adjusted to pH 7.4 with nitric acid was used as make-up liquid. A spike of 40 μg Ge L−1 was used as internal standard and for monitoring the stability of nebulization during CE experiments. The ICP–MS was linked to the interface by a shielded Teflon tube connected directly with the torch.

Fused silica capillaries were used for all CE experiments (Polymicro Technologies, Phoenix, Arizona, USA). Pre-conditioning prior to first usage was performed by flushing the capillary with 10% nitric acid for 10 min followed by flushing for 5 min with ultra-pure water. Final conditioning of the capillary surface was performed by flushing with 1 mol L−1 KOH for another 10 min then flushing for 20 min with the separation buffer. Table 1 shows details of the capillary conditioning procedure performed after each separation to achieve maximum reproducibility.
Table 1.

Capillary conditioning parameters for CE–ORS–ICP–MS

Solution

Concentration (mol L–1)

Time (min)

Method

NH3

1

2

Flush capillary with 100 kPa

KOH

1

4

Flush capillary with 100 kPa

Water

1

Flush capillary with 100 kPa

Buffer

0.02

4

Flush capillary with 100 kPa

Buffer

0.02

2

30 kV

Buffer

0.02

2

Flush capillary with 100 kPa

The auto-sampler of the CE was kept at a constant temperature level of +10 °C with an external cooling system to prevent changes in the viscosity of the sample and the running buffer.

In order to estimate the instrumental detection limits for sulfur and some selected trace elements measured simultaneously, the ICP–MS system was combined with a peltier-cooled quartz spray chamber and a micro-concentric PFA 100 nebulizer (Elemental Scientific, Omaha, Nebraska, USA). All instrumental parameters are summarised in Table 2.
Table 2.

Instrumental parameters

Parameter

CE–ORS–ICP–MS

ORS–ICP–MS

Nebulizer

Cetac CEI 100

PFA Micro flow 100

RF power (W)

1450

1450

Extraction lens (V)

+4 to +6

+4 to +6

Cones

Nickel

Nickel

Sampling depth (mm)

6

6

Carrier gas (L min–1)

1.1

0.85

Make-up gas (L min–1)

0

0.32

OctP bias (V)

–40

–40

QP bias (V)

–3

–3

Xe flow (%)

5

5

Capillary

Fused silica

Capillary length (cm)

70

Capillary i.d. (mm)

0.075

Separation voltage (kV)

30

Column/capillary temperature

+15 °C

Vial table temperature

+10 °C

Sample injection

Hydrodynamically with different volumes

Buffer

Tris-HNO3 20 mmol L–1, pH 7.0–7.4

Sample preparation

Metallothionein preparation

MT solutions of different concentration were prepared gravimetrically by dissolving appropriate amounts of MT in degassed, argon-flushed buffer solution. MT solutions were stored under argon at –18 °C until analysis.

Liver samples

The Elbe bream investigated in this work were obtained from local fishermen working in the region of Gorleben, Lower Saxony, Germany. The fish were killed directly after being caught, washed with tap water and stored in polyethylene (PE) plastic bags at –18° C until further preparation.

Cytosol preparation/extraction of the metallothionein-like proteins

For cytosol preparation about 3 g of liver tissue was transferred into a Potter–Elvehjem homogenisation tube together with 3 mL of cooled (+4 °C), degassed, argon-flushed, Tris-HNO3 buffer (5 mmol L−1, pH 7.4). All sample preparation steps were performed with ice cooling and in an argon atmosphere to prevent oxidation of the sample. The use of reducing agents such as 2-mercaptoethanol or dithiothreitrol was avoided due to their metal-complexing capabilities which could influence the metal composition of the species investigated.

In order to avoid contamination, especially with the elements copper and zinc, all tools (vials, caps, containers bottles etc.) used in sample preparation were cleaned with acid before use.

The liver tissue was homogenised within a few minutes. The homogenate was then centrifuged at 25,000 g for 90 min at +4 °C. The supernatant was separated from the tissue residues and stored under argon at –18 °C until required.

For further sample purification, ultra filtration units with a molecular weight cut-off (MWCO) of 100,000 Da were used to remove high-molecular-weight proteins and particles from the cytosol. Therefore the supernatant was transferred under argon atmosphere into a filtration unit and centrifuged at +4 °C and 8000 g. For the sulfur experiments, pre-concentration of the cytosol was performed. The filtrate obtained in the previous preparation step (2 mL) was transferred into another filtration unit with a MWCO of 3000 Da and centrifuged at 8000 g until a final volume of about 200 μL was achieved.

Element standards

All element standards were prepared by dilution of the stock solution with ultra-pure water to the desired concentration. The solutions were stored in PFA or FEP containers cleaned with acid vapour to minimise contamination. All standards were measured directly after preparation.

Results and discussion

Optimisation of 32S detection

The instrument was tuned without using the collision/reaction mode on a daily basis with a standard tuning solution (1 μg Li, Y, Ce, Tl) to obtain maximum sensitivity and to optimise the nebulization efficiency of the CE–ICP–MS interface.

Under optimised instrumental conditions, which are summarised in Table 2, the octopole reaction system functions as a collision cell. Besides other interferences due to 14N18O+ and 15N16OH+, 16O2+ represents the most abundant polyatomic interference at the mass of 32S+. Table 3 gives an overview of the abundance of the main sulfur isotopes and some interfering molecular ions.
Table 3.

Abundances of sulfur isotopes and relevant polyatomic ions

30 amu

31 amu

32 amu

33 amu

34 amu

35 amu

S+

94.93%

0.73%

4.29%

O2+

99.52%

0.08%

0.4%

O2H+

99.51%

0.09%

0.4%

NO+

99.39%

0.41%

0.2%

For the dissociation of O2+, an energy of 6.66 eV is necessary [22]. The collision energy obtained under the instrumental conditions realised could be calculated according to:
$$ E_{{\rm{cm}}} = {{m_{\rm{k}} } \over {m_{\rm{k}} + m_{\rm{p}} }}E_{{\rm{lab}}} $$
where Ecm represents the collision energy at the centre of mass, mk the mass of the collision gas (Xe 131.29 u), mp the mass of the target ion (O2+ 31.9988 u), and Elab represents the kinetic energy resulting from the potential difference between the extraction lens and the quadrupole entrance. With Elab=44 eV and xenon as cell gas a collision energy of about 35.4 eV is obtained. If there is a collision, O2+ will be dissociated inside the cell.
Figure 1 shows the spectral background of a MilliQ DI water sample in the mass range from m/z 29 to m/z 42 (black spectra). As can be seen, high background count rates were obtained at the masses of 32S, 33S and 34S as a result of the large abundance of interfering polyatomic ions. Under such conditions sensitive determination of sulfur at any of its isotopes is not possible when using quadrupole-based ICP–MS.
Fig. 1.

Spectral background of an acidified MilliQ DI water sample in the mass range from m/z 29 to m/z 42. The black spectra were obtained under normal instrumental conditions. The grey spectra were obtained in the Xe mode by addition of approx. 0.2 mL min−1 Xe into the octopole reaction system

The grey spectrum demonstrates the effect of addition of Xe (5%, approx. 0.2 mL min−1) to the octopole reaction system on the spectral background of the same DI water sample. The spectral background is decreased by about six orders of magnitude enabling the determination of sulfur with quadrupole-based ICP–MS using the most abundant isotope 32S.

Instrumental detection limits

Detection limits for the simultaneous determination of sulfur and some trace elements were derived according to the German standard procedure DIN 32645 [23]. The detection limits for sulfur in acidified MilliQ DI water obtained using the Xe mode were between 1.3 μg L−1 (34S) and 3.2 μg L−1 (32S). For the simultaneously determined trace elements, detection limits in the range 300 ng L−1 (58Ni) to 500 ng L−1 (66Zn, 55Mn) were obtained. It is important to keep in mind that Xe not only has a dramatic effect on reducing the interference due to polyatomic ions, but also reduces the ion transmission of all the measured elements, which results in higher detection limits in comparison with results obtained under standard instrumental conditions. Table 4 shows a compilation of the detection limits obtained using the Xe mode. For information purposes, detection limits obtained using the standard, H2 and He mode are also given [24]. It is not reasonable to measure sulfur using the standard, H2 or He modes, but for the trace elements investigated detection limits ranging from 0.7 ng L−1 (55Mn, H2 mode; 59Co, He mode) to 25 ng L−1 (66Zn, H2 mode) are achievable with ORS–ICP–MS.
Table 4.

Detection limits estimated according to DIN 32645 for sulfur and some simultaneously measured trace elements in an aqueous solution (MilliQ DI water) measured in different ICP–MS modes

Isotope

Detection limits Xe mode (μg L–1)

Detection limits standard mode (ng L–1)

Detection limits H2 mode (ng L–1)

Detection limits He mode (ng L–1)

32S

3.2

34S

1.3

52Cr

0.4

9

9

55Mn

0.5

0.8

0.7

56Fe

0.4

9

58Ni

0.3

9

11

2

59Co

0.4

8

0.7

63Cu

0.4

4

2

66Zn

0.5

25

24

107Ag

0.4

114Cd

0.4

1.2

1

CE–ICP–MS Experiments

Analysis of an MT preparation

Figure 2 shows an electropherogram of a commercially available MT-1 preparation obtained using CE–ORS–ICP–MS under optimised separation and detection conditions (Table 2). Sulfur (32S), copper (63Cu), zinc (64Zn), and cadmium (114Cd) were detected simultaneously. 5% Xe was employed, corresponding to a flow rate of about 0.2 mL min−1 into the octopole reaction system, in order to minimise the background interference at the mass of 32S. The electropherogram shows one dominant and some smaller peak at the mass of 114Cd. Corresponding peaks were also found at the other masses detected. The dominant peak could be assigned to MT-1. By comparison of the migration time with that from other commercially available MT preparations, the less abundant peak with a migration time of around 600 s could be assigned as an MT-2 impurity.
Fig. 2.

CE–ORS–ICP–MS analysis of a commercially available rabbit liver MT-1 preparation (Sigma MT-1, Lot 127H7810) with simultaneous detection of 32S, 63Cu, 64Zn, and 114Cd using the Xe collision mode. The electropherograms are off-set for improved clarity

Quantification of sulfur and cadmium in an MT-1 preparation using external calibration

In order to quantify sulfur and cadmium in an MT sample we suggested the use of an external calibration. One unique property of ICP–MS is that its element detection capability is considered to be independent of the speciation of the element, because the 7000 K argon plasma destroys any type of molecule completely. We suggested the use of cadmium- and sulfur-containing compounds to perform a compound independent calibration, since certified standards for MT are not available. Thiourea and cadmium nitrate tetrahydrate, respectively, were used as sulfur and cadmium compounds. Separation was carried out under slightly modified conditions: The buffer pH was adjusted to 7.0 in order to keep the cadmium stable in solution during electrophoretic separation. Preliminary experiments showed that a change to a more acidic pH influences only the migration properties (separation time decreased slightly due to the reduced EOF) but not the separation efficiency and the resolution of the electropherogram. Figure 3 shows such electropherograms from an MT preparation (Sigma, Lot 19H7812) obtained after separation using CE–ORS–ICP–MS and 20 mmol L−1 Tris-HNO3 buffer adjusted to pH between 6.9 and 8.0, at the mass of 114Cd. As can be seen, the pH particularly influences the migration time of the MT sample due to a reduced EOF as a result of the reduced pH.
Fig. 3.

Electropherograms of a rabbit liver MT preparation (Sigma, Lot 19H7812) separated with CE–ICP–MS using 20 mmol L−1 Tris-HNO3 buffer adjusted to different pH levels ranging from 6.9 to 8.0 measured at the mass of 114Cd . Separation conditions: 30 kV, positive polarity, 20 mbar s (2.2 nL) injection, capillary temperature +15 °C

Figure 4 demonstrates the simultaneous separation of the sulfur and cadmium compounds used for calibration during CE–ORS–ICP–MS experiments. The sample contained 1 mg L−1 Cd (peak b) and 10 mg L−1 S (peak a). Ultra-pure water was used as solvent. Sample injection was performed hydrodynamically for 2 s with 25 mbar pressure giving a sample volume of 5.5 nL. Both element compounds could be separated in one CE experiment in less than 10 min. The peak areas calculated were used for calibration and quantification of cadmium and sulfur in the MT preparation investigated. Linear calibration plots for sulfur (y=537.36x−1274.5, R2=0.9992) and cadmium (y=1367.2x−5370.9, R2=0.9971) were obtained. The calibration data were also used to estimate the detection limits for cadmium and sulfur under CE–ORS–ICP–MS conditions according to the German standard procedure DIN 32645. Detection limits were 1 mg L−1 for cadmium and 15 mg L−1 for sulfur. These values correspond to absolute detection limits of 5.5 pg for Cd and 82.5 pg for S (2.57×10−12 mol S). The detection limits achieved (Table 5) were relatively high due to the small sample volume generally used in CE experiments. Further optimisation of the instrumental sensitivity will therefore be necessary, especially for the investigation of real-world samples. Based on the calibration data, the total sulfur and cadmium contents of a commercially available MT preparation (Sigma MT-1) were calculated. The results are summarised in Table 6. These results were checked using total X-ray fluorescence spectrometry (TXRF) to determine the total element content of the MT sample. The results were comparable, which suggests that this could be a valid way to use species-unspecific element-containing compounds such as thiourea (for sulfur) and cadmium nitrate tetrahydrate (for cadmium) for the quantification of sulfur and cadmium in metallothionein.
Fig. 4.

Simultaneous separation of Cd2+ (cadmium nitrate tetrahydrate) and thiourea in one electrophoretic run. Separation conditions: 30 kV, positive polarity, 50 mbar s (5.5 nL) injection, capillary temperature +15 °C, 20 mmol L−1 Tris-HNO3, pH 7.0. Sample contains 10 mg L−1 sulfur (a) as thiourea and 1 mg L−1 cadmium (b) as cadmium nitrate tetrahydrate. The electropherograms are off-set for improved clarity

Table 5.

Detection limits estimated according to DIN 32645 for sulfur and cadmium obtained under CE–ORS–ICP–MS conditions

Element

Detection limits (mg L–1)

Absolute detection limits (pg)

Sulfur

15

82.5 pg

Cadmium

1

5.5 pg

Table 6.

Quantification of the sulfur and cadmium content of a commercial MT-1 preparation. Concentrations given in mg L–1

TXRF (n=3)

CE–ORS–ICP–MS (n=5)

Sulfur

1082±21

968±50

Cadmium

960±48

1097±21

Element-specific determination of metallothionein-like proteins in liver cytosol samples of Abramis brama L. using CE–ORS–ICP–MS

The investigation of real-world samples containing metallothioneins at natural levels represents a challenging task in metallothionein speciation with CE–ORS–ICP–MS. The main problems are related to the low MT concentration levels found in real samples and the poor detection limits achieved with CE–ICP–MS due to the small sample amounts generally used in CE experiments (normally a few nL). A further problem results in the reduced ion transmission when using Xe as collision gas for sulfur measurement. Therefore, sample clean-up and pre-concentration steps are necessary in order to obtain an adequate sample concentration. Ultra-filtration was used to minimise the interaction of the sample with other chemicals and to avoid the formation of artefacts during sample preparation.

Figure 5 shows the electrophoretic separation of a cleaned and pre-concentrated bream liver cytosol sample using the standard mode (no collision gas). Copper (63Cu), zinc (64Zn) and cadmium (114Cd) were monitored simultaneously during electrophoresis. The electropherogram shows corresponding peak patterns at all detected masses with two major (1 and 2) and some minor peaks. At the masses of 63Cu and 64Zn, in particular, highly abundant peaks were observed. Peak 1 shows incomplete separation, which results in two intensity maxima (Peak 1a, Peak 1b). At the masses of copper and cadmium Peak 1a shows a higher abundance, whereas Peak 1b shows a higher abundance at the mass of zinc. Peak 2 represents the most abundant compound in the cytosol sample investigated. This characteristic peak pattern, which is found at all masses detected, could be a hint of the presence of metallothionein-like proteins.
Fig. 5.

Electrophoretic separation of a bream liver cytosol sample. Element-specific detection of copper (63Cu) zinc (64Zn), and cadmium (114Cd), in the standard mode. Separation conditions: 30 kV, positive polarity, 50 mbar s (5.5 nL) injection, capillary temperature +15 °C, 20 mmol L−1 Tris-HNO3, pH 7.0

Due to the high sulfur content that could be found in metallothionein, the liver cytosol was separated again using the Xe mode to test if sulfur also is present in the cytosol sample. Besides 32S, the trace elements 63Cu, 64Zn and 114Cd were detected simultaneously during electrophoresis. Figures 6a and 6b show the electrophoretic separation and element-specific detection of a bream liver cytosol sample measured using 0.2 mL min−1 Xe as collision gas for interference minimisation at the mass of 32S. The electropherograms show corresponding peak patterns at the masses of sulfur, copper, and zinc and also one highly abundant peak at the mass of sulfur with a small corresponding peak at the other detected masses. Cadmium could not be detected in the Xe mode due to the low abundance of cadmium in the liver sample (Fig. 5, obtained in the standard mode) and the fact that the transmission of all the other elements is reduced when using Xe as collision gas. The occurrence of sulfur in some of the peaks observed also indicates the presence of metallothionein. Further investigations with molecule-specific detection techniques are necessary to finally prove these suggestions.
Fig. 6.

(a) Electrophoretic separation and element-specific detection of a bream liver cytosol sample using 0.2 mL min−1 Xe as collision gas for interference minimisation at the mass of 32S. Separation conditions: 30 kV, positive polarity, 50 mbar s (5.5 nL) injection, capillary temperature +15 °C, 20 mmol L−1 Tris-HNO3, pH 7.0. (b) Enlarged illustration of the 32S electropherogram of Fig. 6a. Electrophoretic separation and element-specific detection of a bream liver cytosol sample using 0.2 mL min−1 Xe as collision gas for interference minimisation at the mass of 32S. Separation conditions: 30 kV, positive polarity, 50 mbar s (5.5 nL) injection, capillary temperature +15 °C, 20 mmol L−1 Tris-HNO3, pH 7.0

Conclusions

ORS–ICP–MS is a suitable technique for the simultaneous determination of sulfur and other trace elements either under normal ICP–MS or under hyphenated conditions. Xenon is an effective collision gas for reducing interference at the masses of sulfur. CE–ORS–ICP–MS allows the simultaneous detection of sulfur, cadmium, copper, and zinc in biologically relevant molecules such as metallothionein or metallothionein-like proteins. External calibration based on the use of sample independent, element-containing compounds is an appropriate technique for the quantification of sulfur and cadmium in an MT-1 preparation. For quantification of real samples a further improvement of the sample-preparation technique is necessary. Comparable results were obtained with TXRF as an independent technique for total element quantification. Further experiments will be necessary to apply the same technique for the quantification of copper and zinc. A promising approach is the use of 2,6-diacetylpyridine-bis(N-methylenepyridiniohydrazone) as a complexing agent for copper and zinc. This enables stable, positively charged complexes under the pH conditions usually used for MT separation to be generated. Finally, CE–ORS–ICP–MS offers enough sensitivity for the separation and element-specific detection of metallothionein-like proteins at a non-induced level in bream liver samples via measurement of their sulfur, copper, zinc and cadmium contents.

Notes

Acknowledgements

The authors would like to thank Simone Griesel and Ulrich Reus for performing the TXRF measurements.

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Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Daniel Pröfrock
    • 1
  • Peter Leonhard
    • 1
  • Andreas Prange
    • 1
  1. 1.GKSS Research Centre GeesthachtInstitute for Coastal ResearchGeesthachtGermany

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