Interference-free determination of ultra-trace levels of Arsenic and Selenium using methyl fluoride as reaction gas in ICP-MS/MS

Department of Analytical Chemistry Research group Atomic and Mass Spectrometry Interference-free determination of ultra-trace levels of Arsenic and Selenium using methyl fluoride as reaction gas in ICP-MS/MS Thesis submitted to obtain the degree of Master of Science in Chemistry by Elisabeth Nissen Academic year 2013-2014 Promoter: Prof. Dr. Frank Vanhaecke Copromoter: Dr. Lieve Balcaen Supervisor: Eduardo Bolea-Fernandez

Acknowledgements/preface During the academic year 2013/2014, Master student Elisabeth Nissen has produced the following Master thesis to obtain the degree of Master of Science in Chemistry. All lab work was performed from September 2013 to May 2014 at the Department of Analytical Chemistry, Atomic and Mass Spectrometry group, Ghent University. It is expected that the reader has a basic knowledge of analytical chemistry. A great thanks to the staff and students at the Department of Analytical Chemistry, Atomic and Mass Spectrometry group, Ghent University, for supplying the instrumentation, facilities and help to execute this Master thesis project. I especially owe PhD student Eduardo Bolea-Fernandez, Dr. Lieve Balcaen and Prof. Dr. Frank Vanhaecke a great thanks, since their help was invaluable in the execution of this Master thesis project.

Abbreviations AAS AFS ICP-AES ICP-MS LOD LOQ SF-ICP-MS Q-ICP-MS ICP-MS/MS CH 3 F DCP MIP IP m/z-ratio ESA TOF Q1 Q2 MS/MS set-up SQ set-up MFC Atomic absorption spectrometry Atomic flourescence spectrometry Inductively coupled plasma-atomic emission spectrometry Inductively coupled plasma-mass spectrometry Limit of detection Limit of quantification Double-focusing sector field ICP-MS Quadrupole ICP-MS Tandem mass spectrometry ICP-MS Methyl fluoride Direct current plasma Microwave induced plasma Ionization potential Mass-to-charge ratio Electrostatic analyzer Time of flight 1 st Quadrupole 2 nd Quadrupole Q1 followed by a collision/reaction cell and Q2 Collision/reaction cell followed by a single quadrupole Mass flow controller

Overview 1. Introduction and aim... 1 2. Theory... 6 2.1 Instrumentation... 6 2.1.1 ICP-MS in general... 6 2.1.2 Interferences in ICP-MS... 13 2.1.3 Triple quadrupole mass spectrometry (ICP-MS/MS)... 14 2.2 Contamination... 18 3. Experimental... 19 3.1 Instrumentation... 19 3.2 Samples and reagents... 21 3.3 Sample preparation... 22 3.3.1 Digestion procedure... 22 3.3.2 Reconstitution procedure for urine... 23 4. Results and discussion... 25 4.1 Optimization of ICP-MS/MS protocol for the determination of As and Se... 25 4.1.1 As... 25 4.1.2 Se... 30 4.2 Optimization of SF-ICP-MS protocol for the determination of As and Se... 36 4.3 Calibration data and limits of detection... 37 4.3.1 As... 37 4.3.2 Se... 39 4.4 Investigation of the improvement in sensitivity by the addition of MeOH... 40 4.4.1 As... 40 4.4.2 Se... 42 4.5 Results obtained for simulated matrices... 44 4.5.1 As... 44 4.5.2 Se... 47 4.6 Results obtained for reference materials - As and Se... 52 5. Conclusion... 56 6. References... 57 7. Appendix... 61

1. Introduction and aim Arsenic (As) and selenium (Se) are two interesting elements to investigate, due to their presence in different sample types e.g., environmental and biological samples, and due to the fact that As is known to be a toxin, whereas Se is an essential element, but it becomes toxic at higher concentrations, while the difference between an appropriate and an excessive concentration is [1, 2] small. Arsenic is a metalloid, which is present in the environment through both a natural route, with an abundance in the earth's crust of 2.5 μg/g, and an anthropogenic route due to industrial, agricultural and mining activities. [3,4] Arsenic is present in both organic and inorganic forms and in various oxidation states (-III, 0, +III, +V). The toxicity is related to its chemical form and oxidation state and inorganic species are known to be more toxic than organic species, with decreasing toxicity for the species as follows: arsenite > arsenate > monomethylarsonate (MMA) > dimethylarsinate (DMA). [1,2,5,6] For a human adult, inorganic As is lethal at an amount of 1-3 mg As/kg, whereas long term exposure to inorganic As has been linked to adverse effects such as skin lesions, cancer, developmental toxicity, cardiovascular diseases, neurotoxicity, abnormal glucose metabolism and diabetes. [5] Selenium is a non-metal, which is also present in the environment naturally, with an abundance in the earth's crust of 0.05 μg/g, but it is also introduced through anthropogenic routes similar to those of As, as a result of agriculture, mining, petrochemical and industrial activities. [7] Selenium also exists in both organic and inorganic forms and in different oxidation states (+VI, +IV, 0, -II), and in the environment, Se species such as Se (IV), Se (VI), dimethylselenide (dmese), dimethyldiselenide (dmedse) and dimethylselenone (dmeseo 2 ) can be found, with organic and inorganic species having different toxicity. As mentioned before, Se is an essential element and it can be linked to biological activities such as antioxidant actions, activation and degradation of thyroid hormones, immunity enhancement, and reduction of colon cancer risk. However for Se, the range between the deficiency level (< 40 μg/day for adults) and the toxic level (> 400 μg/day for adults) is very narrow. A deficit in Se has been linked to some endemic diseases and the exposure to too much Se can lead to selenosis. [2,8,9] Page 1 of 65

Due to As being toxic even at low levels and the fact that for Se the range between toxic or essential is very narrow and at low levels, it is important to be able to determine As and Se at ultra-trace levels with high accuracy. Arsenic and selenium have been investigated earlier using a variety of analytical techniques, such as atomic absorption spectrometry (AAS) [10-12], atomic flourescence spectrometry (AFS) [13-15] and inductively coupled plasma-atomic emission spectrometry (ICP-AES) [16-18]. However, inductively coupled plasma-mass spectrometry (ICP-MS) has to be considered as the technique of choice due to the advantages of very low limits of detection (LOD), a wide linear dynamic range, as well as multi-element and isotopic capabilities. [1,2,19-22] However, the determination of As and Se within a complex matrix by means of ICP-MS is not that straightforward, due to the following reasons: 1) both As and Se have high ionization energies (9.82 ev and 9.75 ev, respectively), which means that they are poorly ionized under normal ICP-MS conditions, and the ionization efficiency is in the order of 52 % and 33 %, respectively [23], leading to poor sensitivity for the elements, and 2) spectral overlap occurs for As and all Se - isotopes (Table 1) as a result of the occurrence of e.g., isobaric, polyatomic and doubly charged ions of the same m/z-ratios, which means that interference-free determination is a challenge. Table 1 As and Se - isotopes with their natural isotopic abundance [24] and the most important isobaric, polyatomic and doubly charged interferences [25,26] (non-restrictive list). Analyte Abundance (%) Isobaric interference 75 As + 100 - Polyatomic interference Doubly charged interference 40 Ar 35 Cl +, 59 Co 16 O +, 36 Ar 38 ArH +, 38 Ar 37 Cl +, 36 Ar 39 K +, 43 Ca 16 O 2 +, 40 Ar 23 Na 12 C +, 12 C 31 P 16 O 2 +, 40 Ca 35 Cl + 150 Nd 2+, 150 Eu 2+, 150 Sm 2+ 74 Se + 0.89 76 Se + 9.37 74 Ge + 37 Cl 37 Cl +, 36 Ar 38 Ar +, 38 Ar 36 S +, 40 Ar 34 S +, 39 K 35 Cl +, 58 Ni 16 O + 148 Sm 2+, 148 Nd 2+ 76 Ge + 40 Ar 36 Ar +, 38 Ar 38 Ar +, 60 Ni 16 O +, 39 K 37 Cl +, 41 K 35 Cl + 152 Sm 2+, 152 Gd 2+ 77 Se + 7.63-38 Ar 39 K +, 61 Ni 16 O +, 59 Co 18 O +, 40 Ar 37 Cl +, 40 Ca 37 Cl + 36 Ar 40 ArH +, 38 Ar 2 H +, 12 C 19 F 14 N 16 O 2 + 154 Sm 2+, 154 Gd 2+ 78 Se + 23.77 78 Kr + 38 Ar 40 Ca +, 62 Ni 16 O +, 41 K 37 Cl +, 40 Ar 38 Ar + 156 Gd 2+, 156 Dy 2+ 80 Se + 49.61 80 Kr + 40 Ar 40 Ca +, 64 Ni 16 O +, 64 Zn 16 O +, 32 S 2 16 O +, 32 S 16 O 3 +, 40 Ar 40 Ar +, 40 Ca 40 Ca +, 160 Gd 2+, 160 Dy 2+ 82 Se + 8.73 82 Kr + 40 Ar 42 Ca +, 34 S 16 O 3 +, 66 Zn 16 O +, 12 C 35 Cl 2 +, 40 Ar 2 H 2 + 164 Dy 2+, 164 Er 2+ In literature, a variety of different approaches have been suggested to deal with these problems. With regard to the problem of poor ionization, several studies have used the addition of organic solvents, such as ethanol or methanol, or organic compounds to induce a signal enhancement of these elements, denoted the carbon effect. The presence of carbon in a sample can influence the ionization conditions in the plasma, the rate at which the aerosol is transported, the nebulization Page 2 of 65

efficiency for the sample and/or the mass load of vapor. The signal enhancement is due to an alteration of the region of maximum ion density in the plasma, an enhancement of the nebulization of the sample and due to a charge transfer reaction from C + species to analyte ions: C + + M M + + C-species (1), where M is the analyte ion. This signal enhancement is especially observed for some species, such as As and Se, so-called hard-to-ionize elements, since carbon has an even higher ionization energy of 11.36 ev, and it can transfer its positive charge according to process (1). [23,27] In literature, studies can be found that report an enhancement of the As signal of 240 % and of the Se signal of 250 % due to the presence of glycerol, and as a results of methane being present an enhancement of 500 % and 300 % for As and Se, respectively, was reported [28], whereas other papers have also reported an enhancement of 150 % for As and Se using methanol [21]. However, if the presence of carbon affects the total intensity of As and Se in a sample, this can also cause a problem, since standards and samples may have a different C-content, and then, external calibration fails. Thus, it is important to use an appropriate internal standard to correct for this enhancement, thus one that also experiences this carbon effect or use standard addition for calibration. [29] In order to overcome the problem of spectral interference, two main approaches have been proposed. 1) Using a quadrupole-based ICP-MS instrument (Q-ICP-MS) equipped with a collision/reaction cell to overcome the problem of spectral interference by chemical resolution, where either the interference is removed or reduced due to collision or reaction with the cell gas or where the analyte reacts with the cell gas and forms a reaction product that can be measured interference-free at another m/z-ratio. [22] By using a collision gas mixture, such as H 2 /He, one can aim at reducing the spectral background at the mass of As and at the masses of the Se - isotopes. However, this will also substantially reduce the transmission of As + and Se + ions. Detection limits of 0.15 μg/l and 0.03 μg/l have been reported for As and Se (via 80 Se), respectively, using this method. [19] Another option is to use a reaction gas such as O 2, in order to convert As + and Se + ions into AsO + and SeO + ions, and measure in the mass-shift mode, where the reaction product can be detected at the new mass (m/z 91 for 75 As + and m/z 90, 92, 93, 94, 96 and 98 for 74 Se +, 76 Se +, 77 Se +, 78 Se +, 80 Se + and 82 Se +, respectively). [30,31] Other typical gases that could be used, are CH 4 and H 2. [32] 2) A second option is to use an ICP-MS instrument equipped with a double-focusing sector field mass analyzer, denoted SF-ICP-MS, where the mass resolution is high enough for interference-free determination. However, this instrument comes at a high purchase price and the use of the high resolution mode results in the loss of sensitivity of 2 orders of magnitude, due to a reduction in the Page 3 of 65

transmission efficiency of the ions, which results in a reduction of the signal intensity. [22] Detection limits of 0.004 μg/l [33] and 0.004 μg/l [34] have been reported for As and Se (via 82 Se), respectively, using this technique. Typically SF-ICP-MS is the method of choice for ultra-trace level determination of As and Se, since it offers the best limits of detection. In 2013, a new generation of quadrupole-based ICP-MS instrumentation was introduced. This new generation of instruments is based on a triple quadrupole (ICP-QQQ) set-up, where an octopolebased collision/reaction cell is placed in-between two quadrupole mass analyzers. This allows for the operation of the ICP-MS/MS instrument in MS/MS mode, which should be able to deal better with spectral overlap. The improvement is mainly due to the introduction of the first quadrupole before the collision/reaction cell, which only lets the analyte and other on-mass ions pass. Thus, the reactions in the cell are more under control, and additionally, the new set-up makes it possible to perform a product ion scan, which can be used to easily identify which products are formed. [22] This opens up the possibility to potentially be able to match the detection limits obtained using SF-ICP- MS (or maybe even obtain better detection limits). As an example, in literature it can be found, that in a study on the determination of titanium in blood, using ICP-MS/MS and NH 3 /He as reaction gas, instrumental detection limits were found equal to those obtained by SF-ICP-MS. [35] Typical gases used for ICP-MS/MS are H 2, He, NH 3, O 2 or a mixture of these. However in this project, a rather unconventional reaction gas, methyl fluoride (CH 3 F), is investigated for the use in determining As and Se at ultra-trace levels. In the literature, it can be found that CH 3 F has been used before as reaction gas, e.g., in determining the 87 Sr/ 86 Sr isotope ratio in magmatic rocks, which suffers from the isobaric interference of 87 Rb + and good results were obtained [36] or in order to conduct isotope-dilution determination of vanadium ( 50 V +, 51 V + ), where 50 V + suffers from isobaric interferences from 50 Ti + and 50 Cr +, and it was shown that with a mixture of CH 3 F and NH 3 as reaction gas, it was possible to reduce the isobaric interferences [37]. In a study conducted by Xiang Zhao et al, it was investigated which kind of reactions CH 3 F had with atomic transition - metal and main - group cations using an ICP-SIFT tandem mass spectrometer. It was found that CH 3 F mainly reacts in 5 ways, listed below. [38] Page 4 of 65

M + +CH 3 F M + CH 3 F (1) - Molecular addition MF + + CH 3 MCH + 2 + HF MCHF + + H 2 CH 2 F + + MH (2) - F atom transfer (3) - HF elimination (4) - Dehydrogenation (5) - Hydride transfer In this project, the capabilities of the reaction gas CH 3 F to resolve the spectral overlaps that As and Se suffer from was investigated. The main reaction path between the reaction gas and the elements of interest was determined and a selective and sensitive method for As and for Se determination was developed. These methods were evaluated in terms of sensitivity and limit of detection, and compared to other modes of operation using no gas, He and single quadrupole mode and other types of instrumentation, such as SF-ICP-MS. Additionally, validation was performed by use of simulated matrices and by measurement of a diversity of certified reference materials. Page 5 of 65

2. Theory In this part, the theory behind the instrumentation and contamination will be discussed. Firstly, a general description of the technique inductively coupled plasma-mass spectrometry (ICP-MS) will be given, including its advantages and disadvantages. Subsequently a more detailed description of the tandem mass spectrometer will be described and finally, the issues of contamination will be discussed. 2.1 Instrumentation 2.1.1 ICP-MS in general ICP-MS is a mass spectrometric technique, which can be used to identify and quantify trace elements in samples. The advantages of ICP-MS are its speed of analysis (high sample throughput), low detection limits, and its isotopic and multi-element capabilities. In an ICP-MS instrument, ions are formed in an inductively coupled plasma and these ions are then analyzed using MS. Figure 1 shows a (6) Ion detector schematic overview of a basic ICP-MS instrument, starting with a sample introduction system (1), comprising of a nebulizer and a spray chamber, followed by an ICP torch (2). Hereafter there is an interface region (3) with the sampling and the skimmer cone, which is followed by an ion-focusing system (4). After this, there is a mass separation device (5) and finally, there is an ion detector (6). Region 1 and 2 are under atmospheric pressure, whereas 3-6 are kept under vacuum. [39] (5) Mass separation device Turbo molecular pump (4) Turbo molecular pump Ion optics Figure 1 Schematic overview of an ICP-MS instrument: (1) Sample introduction system, (2) ICP torch, (3) Interface region, (4) Ionfocusing system, (5) Mass separation device and (6) Ion detector. Modification of figure from [39]. (3) Interface Mechanical pump (2) ICP Torch (1) Spray chamber Nebulizer RF power supply 2.1.1.1 Sample introduction system The purpose of the sample introduction system ((1) on figure 1) is, as the name suggests, to introduce a representative part of the sample into the system. For liquid samples, this can be split into two events - aerosol formation and droplet selection. A liquid sample is transported to the nebulizer using a peristaltic pump or by spontaneous nebulization due to a pressure drop in the nebulizer, created by leading the nebulizer gas flow through a narrow hole at the tip of the nebulizer, which is also known as the venturi effect. When the sample reaches the nebulizer, Page 6 of 65

pneumatic action of the gas flow breaks the sample into a fine aerosol by mechanical force. The typical gas used is argon. The nebulizer can have different designs, such as concentric, microconcentric, microflow and cross-flow, where the choice of nebulizer can be done based on the sample under investigation. In this project, the concentric nebulizer is used and a schematic overview of the concentric nebulizer can be seen in figure 2, together with the aerosol generation. [39] Nozzle Shoulder 6 mm Shell or barrel Capillary Seal Radius Annulus Capillary Sample passage 4 mm Uptake tube (liquid input) Nozzle end surface Sidearm Maria (gas input) A) B) Figure 2 A) Schematic overview of the concentric nebulizer and B) An aerosol generated by this nebulizer. [39] This particular design of nebulizer gives good stability and sensitivity, particularly when analyzing clean solutions, however, since the capillary is quite narrow, problems with blockage can be an issue. [39] After the nebulization process, the tiny droplets enter the spray chamber, where the droplet selection Central tube Drain tube Spray chamber Sample aerosol Plasma torch sample injector Figure 3 Schematic overview of the Scotttype double-pass spray chamber. [39] takes place. Only the smallest droplets are sent off to the plasma source for further analysis in order to limit the solvent load of the plasma. Two typical designs are the Scott-type double-pass and the cyclonic spray chamber, however only the Scott-type double-pass spray chamber will be discussed here. In the double-pass design (Figure 3) the aerosol from the nebulizer is guided into a central tube and the droplets then pass through the entire length of the tube, where, due to gravitational forces, the larger droplets (larger than ~ 10 μm in diameter), will drop out and they are removed through a drain tube, which is located at the end of the spray chamber. The smaller droplets (< 10 μm in diameter) will however continue by passing between the central tube and the outer wall due to a positive Page 7 of 65

pressure and from there, they go to the plasma source. This selection of only the smaller droplets can however also be seen as a weak point of the instrumentation, since only 2-5 % of the sample is introduced into the plasma source. A second feature of the spray chamber is to smoothen out the nebulization pulses produced by the peristaltic pump if used. Furthermore, the spray chamber can be externally cooled in order to reduce the introduction of solvent going to the plasma source, which is often required when dealing with organic solvents. [39] 2.1.1.2 Plasma source and ion formation There are different types of plasma sources, with inductively coupled plasma (ICP) being the most common type of plasma source today, but other plasma sources exist, like the direct current plasma (DCP) and the microwave-induced plasma (MIP). In this project, the ICP was used and thus, it is the only one discussed. [39] In the plasma source ((2) on figure 1), which consists of the plasma torch, an RF coil and a power supply, the sample aerosol emerging from the sample introduction system is converted into ions. A schematic overview of the ICP torch can be seen in figure 4. The plasma torch comprises of three concentric tubes - an outer tube, a middle tube and a sample injector. Between the outer and middle tube the gas, which is used as plasma and cool gas, flows, whereas the gas that flows between the middle tube and the sample injector, known as the auxiliary gas, is used to optimize the position of the plasma. Finally, a third gas, known as the nebulizer/carrier gas, transports the sample from the sample introduction system to the torch, where the gas flow physically makes a hole in the ICP. [39,40] Carrier gas with sample aerosol Plasma gas or cool gas Load coil Figure 4 Schematic overview of the ICP Torch. [40] However, before ionization of the sample can occur, the inductively coupled plasma has to be formed. This is done by a load coil to which an RF power is applied, which results in an alternating current that oscillates within the coil at a frequency of either 27.12 or 40.68 MHz. This generates a strong time-dependent electromagnetic field in the top part of the torch, and by means of a high- Torch Time-dependent magnetic field Electrons moving along circular paths ICP Page 8 of 65

voltage spark to the plasma gas flowing through the torch, some electrons are removed from their argon atoms. These electrons, which are forced to move according to circular paths, lead to a spiraling motion. This motion causes the electrons to have high energy and upon collision with other argon atoms, these can be ionized: Ar + e - Ar + + 2e -, resulting in the removal of more electrons. This results in a collision-induced ionization of argon, which will continue in a chain reaction. As a result, a gas comprised of argon atoms, argon ions and electrons is obtained, which is also known as an inductively coupled plasma. Additionally, the coil used in ICP-MS is grounded so that the ICP potential remains close to zero, in order to avoid the formation of a secondary discharge between the plasma and the grounded interface cone. [39,40] A) 6500 K 6000 K 7500 K 8000 K Preheating zone B) Ions Atoms Gas Solid Liquid Normal analytical zone 10,000 K Initial radiation zone Ionization Atomization Vapirization Drying Figure 5 A) The different temperature zones in the plasma, and B) The transformation the sample undergoes in the plasma from liquid to ions. [39] Ion formation in the ICP-MS can be expressed as the generation of positively charged ions using a high temperature plasma discharge. In the plasma, where different heating zones are present, as shown in figure 5A, the sample undergoes a transformation from a liquid aerosol to ions, as shown in figure 5B. First, the droplet is dried, forming a solid particle, the solid then transforms into gas form due to vaporization and continues to a ground-state atom due to atomization. Finally, it reaches the analytical zone of the plasma, where the temperature is 6000-7000 K, where, due to collisions with energetic electrons, excited Ar atoms or Ar ions, the atoms are converted into ions. The extent of positive ion formation is dependent on the ionization potential (IP) of the element and the lower the IP, the easier it is to ionize the atom. After ionization, the ions continue to the interface region. It should be mentioned that it is possible to work with different diameters of the central tube of the torch and especially when using volatile organic solvents it is preferred to work with smaller diameters to avoid extinguishing the plasma. [39] Page 9 of 65

2.1.1.3 Interface region The interface region ((3) in figure 1) bridges the pressure difference between the plasma source (~ 1010 mbar) and the mass spectrometer analysis region (10-6 mbar). [39] The interface comprises of 2 metallic cones (Figure 6) - the sampling cone has a central opening with a diameter of 0.8-1.2 mm, while the hole of the skimmer cone has a smaller diameter of 0.4-0.8 mm. The region between the cones is kept under a vacuum of 1-3 mbar and are conventionally made of nickel, but can also be made of e.g., platinum, which is a lot more resistant to corrosive liquids. [39] After the ions are generated in the plasma source, only a part of the ICP is guided into the interface region, where supersonic expansion occurs between the sampling and skimmer cone due to a lower pressure, which results in the composition of the plasma being frozen. [40] After this, the central beam departs through the skimmer cone and is directed to the ion-focusing system. [39] Skimmer cone Ion optics ~2 Torr vacuum Sample cone RF coil Plasma torch Figure 6 Schematic overview of the interface region. [39] 2.1.1.4 Ion-focusing system As the ICP-MS is set up for detection of positive ions, an ion-focusing system ((4) in figure 1) is needed for removal of neutral species, negative ions and electrons, and to focus the ion beam into the mass analyzer. After the beam emerges from the skimmer cone, the positively charged ion beam is generated, however, due to the net charge being positive, the ions now repel each other and the center of the beam will mainly consist of ions with high massto-charge ratio (m/z-ratio), whereas ions of low m/zratio will be driven to the outside. The degree to which this occurs will depend on the kinetic energy of the ions, where transmittance decreases with decreasing kinetic energy. This is known as the phenomenon of space-charge effect (Figure 7). [39] Ion optics Heavy mass ions Ion flow Medium mass ions Light mass ions Interface Figure 7 Illustration of the space-charge effect produced due to repulsion between ions. [39] Page 10 of 65

In order to hold/bringback the ions in/to the center of the ion beam, ion optics are used, which are a combination of one or more ion lenses to which a voltage is applied and this region is normally kept under a vacuum of 10-3 mbar. The lens(es) only select(s) the positive ions, whereas neutral species, electrons and negative ions are avoided using a physical barrier. Generally, three approaches can be used as barrier: 1) Photon stop, where a metal disk is placed after the skimmer cone and all species that do not change trajectory due to influence of electrostatics hit the disk, 2) Off-axis detector, and 3) Ion mirror, where the ion beam is bended over 90 into the mass analyzer using electrostatics. [39] 2.1.1.5 Mass analyzers From the ion-focusing system, the ion beam enters the mass analyzer and the purpose of the mass analyzer ((5) in figure 1) is to separate the ions as a function of their m/z-ratio and thereby separating the ions, which are significant for the analysis, from everything else, such as the ions derived from the matrix, other elements, the solvent etc.. This can be achieved using different types of mass spectrometers, such as quadrupole mass filter, double-focusing sector field or a time-offlight mass spectrometer. The different devices have different advantages and disadvantages concerning mass resolution, speed and cost, and the device used should be chosen on the basis of the problem under investigation. The mass analyzer is kept under a vacuum of 10-6 mbar and it is positioned between the ion-focusing system and the detector. [39] Quadrupole mass filter When using a quadrupole mass filter, separation is done using four rods (Figure 8), where on each pair of rods a direct current (DC) component, +U or -U and a radio frequency (AC) component is placed with the two pairs of rods having a DC component with opposite sign and an AC component with a phase difference of 180. [40] This results in a band pass filter, where only ions of selected m/z-ratio are allowed to pass through the set-up to the detector. This is due to the electrostatics steering the ions on a ions entrance slit quadrupole rods unstable path stable path exit slit to detector Figure 8 Illustration of the basic principle of the quadrupole mass filter as mass analyzer. [40] trajectory that leads them all the way through, while ions of other m/z-ratios will collide with the Page 11 of 65

rods. By changing the voltages applied to the quadrupole rods, different m/z-ratios can be monitored and a scan of masses from 0-300 amu can be obtained. [39] The advantages of this technique are its simplicity, the lower purchase price, its high scanning speed and its lower sensitivity to differences in kinetic energy of the ions that enter the mass analyzer. However a drawback of this technique is its unit mass resolution. [40] Double-focusing sector field This mass spectrometer comprises of two analyzers: an electromagnetic and an electrostatic analyzer (ESA) and the way of combining both sectors leads to different set-ups: a standard (ESA first) or a reverse Nier-Johnson geometry (ESA last) or a Mattauch-Herzog set-up. Figure 9 shows an example of a set-up, in this case a reverse Nier-Johnson geometry. In the magnetic sector, the Magnetic sector Ion source ions are separated as a function of their m/z-ratio, and the electrical sector focuses the ions on the basis of their kinetic energy. The double focusing effect refers to both focusing of the energy and of the direction, which means that ions of same m/z-ratio, but a difference in kinetic energy and/or direction are focused in one point. [39,40] The main advantage of this set-up is its high mass resolution of R max 10.000, while the scanning speed is still relatively high, although it takes a bit longer to generate a full mass spectrum than when using a quadrupole mass filter and high costs are involved. Another disadvantage is that you lose sensitivity by increasing the resolution, which amounts to a factor of 10 per resolution mode. [40] Time-of-flight Ion acceleration Detector Figure 9 Schematic overview of a reverse Nier-Johnson geometry for doublefocusing sector field. [40] In the time-of-flight (TOF) mass spectrometer, ions, in packages, are accelerated over a difference in potential and enter a flight tube, + V Electrostatic sector + V Repeller ICP Acceleration Flight tube Sampling cone & skimmer Extraction region Page 12 of 65 Figure 10 Schematic representation of the time-of-flight mass analyzer. [40]

where the distance traveled is proportional to the mass and the amount of kinetic energy. By using a reflectron, where ions with higher kinetic energy go in deeper, the ions with the same m/z-ratio are focused in the same point even if they have a different kinetic energy. Figure 10 shows a schematic representation of the TOF. With the TOF, unit mass resolution can be obtained, but the speed of analysis is very fast and a full mass spectrum can be measured in only 0.033 ms. [39,40] 2.1.1.6 Ion detectors After the mass analyzer, the ions reach the ion detector, which transforms the incident ions into electrical pulses. The pulses are counted and the number of the pulses is proportional to the amount of analyte ions, which is present in the sample. Different types of detectors can be used, such as electron multipliers - continuous or discrete dynode - and faraday cups. Here only the discrete dynode electron multiplier will be discussed. [39] In the discrete dynode electron multiplier (Figure 11), the ion hits the inner surface and an electron is released. A potential difference is present between two subsequent dynodes, which leads to the acceleration of the electrons towards the end of the dynode and as the electron collides with the inner surface, additional electrons are produced, Ions in leading to a multiplication effect of the electrons. Thus one ion reaching the detector leads to 10 7-10 8 electrons. It is possible to use the detector in both pulse and analog mode in order to allow determination of both low and high concentrations in the same sample. Due to the fact that detection of one ion takes time and during this time no other ions can be detected, a phenomenon called detection deadtime, τ, occurs and it is normally in the range of 5-100 ns. However, when the value of τ is known, the software can automatically correct for this. [39,40] Signal out (X10 6 ) + 2 KV Figure 11 Schematic overview of the electron multiplier with discrete dynodes. [40] 2.1.2 Interferences in ICP-MS The interference in ICP-MS can be split into two different categories - physical and chemical interferences, which is due to volatility, viscosity, surface tension, density and/or sample transport; and spectral and non-spectral interferences. [39] Page 13 of 65

Spectral interference arises when two or more ions have the same or very similar m/z-ratio. The interference can be in the form of an isobaric overlap, e.g., 40 Ar + / 40 Ca +, polyatomic ions comprising of elements from the plasma gas, matrix, solvent or air, leading to argon-containing polyatomic ions, e.g., 40 ArC + / 52 Cr +, oxide and hydroxide ions, e.g., 32 S 16 O + / 48 Ti +, 136 Ba 16 O 1 H + / 153 Eu +, or other types, e.g., 28 Si 35 Cl + / 63 Cu +, or from doubly charged ions, e.g., 48 Ca 2+ / 24 Mg +. The degree of interference will depend on both the concentration of the interfering element and the analyte element affected by the interference. Some spectral overlaps can be overcome or avoided by using an appropriate procedure for digestion of the samples, aerosol desolvation, by separating the element from the matrix before analysis, by using another sample introduction system, by using mathematical interference correction equations or by using cool/cold plasma technology. Another mean of overcoming spectral overlap is using a mass analyzer with a higher mass resolution than just unit mass resolution, but this also involves higher costs. Alternatively one can also use a collision/reaction cell, where a collision or reaction gas is used to overcome the interference. The principles of the collision/reaction cell will be described in more detail in section 2.1.3. [39,40] Furthermore, non-spectral interferences, whereby the matrix induces a signal suppression and/or enhancement, can be compensated for by using sample dilution, internal standardization, standard addition or a combination of these or isotope dilution. With regard to internal standardization, it is important to choose an internal standard that is not present in the sample already and one that is close to the analyte of interest both in mass number and in ionization potential in order to obtain a good correction. Additionally, the internal standard is also used to correct for instrumental instability and/or signal drift. [41] 2.1.3 Triple quadrupole mass spectrometry (ICP-MS/MS) Typically quadrupoles as mass analyzers are not very good when it comes to dealing with spectral interferences, since they only offer unit mass resolution. That is why nowadays a collision/reaction cell is often placed in front of the quadrupole mass spectrometer, to deal with interferences on the basis of chemical resolution. [39] In the field of ICP-MS collision/reaction cell technology has proven to be a useful technique to remove or avoid spectral interferences by either collision or reaction of the analyte and/or interfering ions with a gas. The basic principle of a collision/reaction cell is that in a cell, which is a Page 14 of 65

multipole - quadrupole, hexapole or octapole -, a collision/reaction gas is added. Typically the gases used are He as collision gas and H 2, O 2, NH 3 or CH 4 as reaction gases. Depending on the ions involved, the gas will either act as a collision gas or as a reaction gas, resulting in processes such as transfer of a proton, transfer of a hydrogen atom, molecular association reactions, fragmentation due to collision, loss in kinetic energy due to collision and focusing due to collision. By choosing the gas wisely, it is possible to remove most spectral interferences. In case a chemical reaction is exploited, either the interfering ions, whereby harmless non-interfering ions are produced, or the analyte ions, in order to convert them to other ions, which can be determined interference-free at another m/z-ratio, can be involved in reaction. After the collision/reaction cell, the ions continue to a quadrupole mass analyzer, which separates the ions according to their m/z-ratio. However, it is important to mention, that along with the reaction of interest, other undesirable collisions/reactions take place, leading to the production of unwanted interfering ions. These can be discarded by either discrimination based on kinetic energy or by discrimination based on mass. [39] Discrimination by kinetic energy (Figure 12) has the purpose of discriminating between unwanted product ions and the analyte ions on the basis of their kinetic energy. This can be done by positioning a potential barrier at the end of the collision cell, whereby ions with less energy than the potential of the barrier will not be able to pass. In this way, the reaction product ions, which have a lower kinetic energy, will not be able to cross the energy barrier, while the more energetic analyte ions will be transmitted to the mass spectrometer. Kinetic energy discrimination can be also be used to discriminate against polyatomic ions. In this case, typically non-reactive gases are used, such as He or Xe. Polyatomic ions are larger, collide more, loose more energy and can thus be prevented from entering the mass spectrometer. [39,42] Quadrupole Cell Pre-cell To detector Energy barrier From plasma Collision/reaction gas atom or molecule Analyte Ion M + - small collision cross-section Polyatomic species e.g., ArX + - large collision cross-section Figure 12 Overview of the principles behind kinetic energy discrimination. [39] Discrimination by mass has the purpose of discriminating against products produced via unwanted reactions on the basis of their mass. For this purpose, the quadrupole offers better capabilities then the higher order multipoles, due to less diffuse stability boundaries, whereby it is possible to Page 15 of 65

selectively filter masses out. As a result, very reactive gases, such as H 2, O 2, CH 4 or NH 3, can be added in the cell. This is beneficial since these gases tend to be better at reducing some inferences, owing to more ion-molecule reactions occurring. Meanwhile, most of the reaction by-products that could lead to new interfering ions are discarded in the quadrupole by the bandpass filter. This is known as dynamic reaction cell technology. [39,43] Sampling cone Ion lens Detector Tandem mass spectrometry, in the form of ICP-MS/MS, takes the concept of the collision/reaction cell a step further. In the MS/MS set-up (Figure 13), the mass analyzer consists of a quadrupole (Q1) followed by a collision/reaction cell, which Plasma Skimmer cone typically is a multipole - in the case of the Agilent 8800 ICP-QQQ it is an octopole, followed by a second quadrupole (Q2). The purpose of the first quadrupole is to operate as a mass filter, and only let the target analyte mass through, thereby preventing all off-mass ions from entering the cel1. This results in a more efficient removal of interferences due to less unwanted species being present in the cell. The cell can function both in a collision and in a reaction mode, as described above, depending on the choice of gas and instrumental settings. Q2 then functions as a second mass filter and can either be used in on-mass mode, where the unreactive analyte can be measured at its original mass, which is now interference-free due to the reaction of the interfering ions in the cell with the reaction gas, or in mass-shift mode, where the analyte, as a result of reactions with the reaction gas, has moved to a new mass, where interference-free determination is possible. Additionally, the ICP- MS/MS can be operated in both single quadrupole (SQ) mode and in MS/MS mode, where single quadrupole mode refers to an instrumental setting of the instrument, where only the collision/reaction cell and the second quadrupole, Q2, are used, and the bandpass of Q1 is operating in "fully open" mass width. The MS/MS mode refers to the instrumental setting of the instrument where both quadrupoles, Q1 and Q2 are used as mass filters. [22,35] Q-pole (Q1) Q-pole (Q2) Octopole reaction system Figure 13 Schematic overview of the MS/MS set-up in relation to the other compartments of the ICP-MS. [44] Page 16 of 65

42 Ca/ 48 Ti + 36 Ar 12 C + 33 S + / 34 S + 16 + O 2 32 S + 42 Ca/ 48 Ti + 36 Ar 12 C + 33 S + / 34 S + O 2 Reaction gas 16 O 2 + 32 S 16 O + 1 st Quad (Q1) Rejects ALL masses except analyte ( 32 S) and on-mass interferences ( 16 O 2) ORS 3 Cell Converts S + to SO + product ion 2 nd Quad (Q2) Set at Q1 + 16 amu Rejects all cell formed ions apart from 32 S 16 O + Figure 14 shows a schematic representation of the ICP-MS/MS system functioning in the MS/MS mode for measuring 32 S in mass-shift mode in the form of 32 S 16 O +. [22] Figure 14 shows an example of MS/MS being used in mass-shift mode in order to measure 32 S in the form of 32 S 16 O +. Here, Q1 is set at an m/z-ratio of 32, so only species with an m/z-ratio of 32 are transmitted to the cell. In the reaction cell, the reaction gas O 2 is used to convert 32 S + into 32 S 16 O +. If only 32 S + reacts with O 2 in the reaction cell, 32 S 16 O + will be the only ion transmitted to the detector when Q2 is set at an m/z-ratio of 48. The other species will be rejected since they are off-mass. [22] Additionally, different scan modes are possible in the MS/MS mode, which can be used for research and method development, such as product ion scan, neutral gain/loss scan and precursor ion scan. In a product ion scan, the m/z-ratio is fixed via Q1 and with Q2, the full mass range is scanned. This mode offers the possibility to evaluate if the analyte reacts or not, and if it reacts, to identify which reaction product ion is the most abundant one, thereby aiding in the interpretation of what reactions are taking place in the reaction cell. In a neutral gain/loss scan, Q1 and Q2 scan over the mass range with a fixed mass difference. This mode can be used to only observe one transition, e.g., a fixed mass difference of 16, which is the addition of an oxygen atom, and the original isotopic pattern can be preserved while eliminating all other oxygen isotopes. In a precursor ion scan, the mass range is scanned using Q1 and the Q2 m/z-ratio is fixed. This mode can be used for identifying the origin of a reaction product determined at a specific m/z-ratio. [22,42,45] The MS/MS set-up described here offers multiple advantages over the single quadrupole set-up (SQ), which was the predecessor. The SQ only consists of the collision/reaction cell followed by a quadrupole. Thus due to not having a quadrupole before the cell, all ions present in the ion beam enter the cell and an ion, that was not leading to spectral overlap with the analyte before, can now, due to reaction with the gas, become a new interference for the non-reactive analyte ion. This is avoided in the MS/MS set-up by Q1, which rejects the parent ion and thereby does not let it reach Page 17 of 65

the cell, and no new interference is formed. Additionally, in the SQ configuration, the mass-shift method does not work if there are unreactive ions present originally at the new mass of the analyte after reaction. Again this is not a problem in the MS/MS set-up, since these ions are rejected via Q1. Thus, the ICP-MS/MS offers a large improvement in the performance of the reaction mode, and an improvement is also seen in the collision mode, where the removal of interferences is also improved due to the cell conditions remaining consistent even if the sample matrix varies. [22,45] 2.2 Contamination In any analysis, it is important to keep contamination to a minimum, but especially when working with trace elements (concentrations < 100 μg/g) and ultra-trace elements (concentrations < 0.001 μg/g), it is crucial to keep contamination to a minimum during sample preparation and analysis. [46] This is due to the fact that any contamination can lead to a large contribution to the measured concentration. However, by using a method blank, which contains all components except the analyte and has been through all the same steps as the sample, it is possible to quantify the contribution of contamination from reagents and the procedure used to prepare the sample in general. [47] Additionally, reagents can also be chosen which have a high purity in order to minimize the contamination, and the equipment used for sample preparation can also be carefully chosen. This can be done by choosing materials that contain low levels of the elements of interest and before use they can be pre-cleaned to remove additional contamination. Furthermore, working in a clean environment can additionally reduce the amount or risk of contamination. [39] Page 18 of 65

3. Experimental 3.1 Instrumentation The instrument used in this work, to carry out all measurements, is an Agilent 8800 triple quadrupole ICP-MS instrument (ICP-QQQ/Agilent technologies, Japan). The instrument is equipped with an introduction system, comprising of a concentric nebulizer followed by a Scotttype double-pass spray chamber. This is then followed by an ICP torch with a central tube with an inner diameter of 2.5 mm, the sampling and the skimmer cone, which are made from nickel, and the mass separation device, which comprises of two quadrupole mass analyzers with an octopole-based collision/reaction cell fitted inbetween, and this is then followed by the ion detector in the form of a discrete dynode electron multiplier. Figure 15 shows a picture of the instrument used. The instrument can be operated in the "vented mode" (no gas in the cell) or the cell can be pressurized with a collision gas (e.g., He) or a reaction gas (e.g., H 2, O 2, NH 3 ) or a mixture of both. However, in this project a rather unconventional reaction gas was used, CH 3 F/He in a 10/90 % mixture, which further on will be denoted CH 3 F. The gas was introduced via the 4 th gas inlet, where the gas flow rate is controlled by a mass flow controller (MFC), which is calibrated for O 2, and allows a flow of 0-100 %, which is equivalent to 0-1 ml/min O 2. Another option would have been to attach the reaction gas to the 3 rd gas inlet, where higher flows are possible, but due the limitation of the set-up of the instrument, that always mixes 1 ml/min He with the gas introduced via the 3 rd beneficial. Figure 15 Agilent 8800 ICP-QQQ, with zooms of the introduction system, the octopole collision/ reaction cell and the electron multiplier. Modification of figure from [48]. gas inlet, this would not be In this project, CH 3 F as a reaction gas in the collision/reaction cell, is evaluated for its possibilities in interference-free determination of 75 As and of the Se - isotopes 77 Se, 78 Se and 80 Se in different types of simulated matrices and in certified reference materials, and the performance is compared to Page 19 of 65

modes using He and no gas in the collision/reaction cell. Additionally, the performance of the methods developed for the ICP-MS/MS are also compared with that of another type of instrumentation, the Thermo Element XR sector-field ICP-MS instrument (Thermo Scientific, Germany), which was - until now - the method of choice for the determination of low levels of As and Se, since it offers the lowest detection limits. The most important instrument settings and parameters used in the experiments in MS/MS mode and SQ mode for the Agilent 8800 ICP-QQQ are listed in table 2 for As and Se and a full list of the instrument settings and parameters can be found in appendix 1. Table 3 lists the conditions used for the Thermo element XR SF-ICP-MS instrument. Table 2 Instrument settings for the Agilent 8800 ICP-QQQ instrument when measuring As and Se. Agilent 8800 Analyte: As Analyte: Se CH 3F No gas He CH 3F He Scan type MS/MS or SQ MS/MS MS/MS MS/MS or SQ MS/MS Plasma mode Low matrix Low matrix RF power 1550 W 1550 W Carrier gas flow rate 1.18 L/min 1.18 L/min 1.05 L/min 1.13 L/min Reaction gas flow rate 72 % - 4.0 ml/min He 100 % 4 ml/min He Q1 Bias -2.0 V -1.0 V 0.0 V -1.0 V 0.0 V Octopole Bias -4.1 V -4.1 V -18.0 V -4.1 V -18.0 V Energy discrimination -8.4 V -8.4 V 5.0 V -8.4 V 5.0 V Q2 axis offset -0.01-0.01 Q2 Bias -12.5 V -12.5 V -13.0 V -12.5 V -13.0 V 77 91 91 77 77 75 89 89 75 75 75 75 78 92 92 78 78 Q1 Q2 78 78 78 78 78 78 78 80 94 94 80 80 125 125 125 125 125 125 125 Wait time offset 2 ms 2 ms Nr. Replicates 10 10 Nr. sweep replicates 100 100 Integration time 1 s 1 s Table 3 Instrument settings for the Thermo Element XR SF-ICP-MS instrument when measuring As and Se. Element XR Scan type EScan Resolution High RF power 1200 W Carrier gas flow rate 0.975 L/min Mass window 100 % Search window 70 % Integration window 60 % Sample time 0.01 s Sample/peak 20 Nuclides monitored 75 As, 77 Se, 78 Se, 125 Te Total analysis time / sample 180 s Page 20 of 65

3.2 Samples and reagents Reagents and solvents: For sample preparation, high purity reagents were used. Water (H 2 O) was purified by a Direct Q-3 Milli-Q system (Millipore, USA) and HNO 3 (14 M, pro analysis, Chemlab, Belgium) was prepared from pro-analysis grade nitric acid by purifying it using a Teflon sub-boiling distillation set-up (Cupola still, PicoTrace ). Other reagents and solvents used were MeOH (25 M, Chromasolv, Sigma Aldrich, Germany), H 2 O 2 (9.8 M, Trace select, Fluka, Belgium) and HF (28 M, trace analysis, Fisher Chemicals, Great Britain). Elemental standard solutions: Elemental standard solutions of 1 g/l of As, Te, Nd and Zr (PlasmaCal, SCP Science, Canada), 1 g/l of Ca, Y, Ge and Zn (Inorganic Ventures, The Netherlands), 1 g/l of Mo, Gd and Sm (Alfa Aesar, Germany), and 10 g/l of Se (Aldrich chemical company inc., USA) were used. From these elemental standard solutions and from HCl (12 M, trace analysis, Chemlab, Belgium) standard solutions used for optimization, internal standardization, calibration and simulated matrix experiments were prepared by dilution with 0.14 M HNO 3. Selenium and tellurium were used as internal standards for measuring As and for the measurement of Se, only Te was used as internal standard. An external calibration curve was prepared by preparing a set of 5 standard solutions in the concentration range 0-5 μg/l of As and of Se, respectively. Samples: The methods for the determination of As and of Se were validated using the certified reference materials listed in table 4. These samples were analyzed following the procedure described under sample preparation, section 3.3. Page 21 of 65

Table 4 List of certified reference materials investigated along with their certified values for As and Se. NBS SRM 1575 Pine needles 0.21 ± 0.04 NBS SRM 1573 Tomato leaves 0.27 ± 0.05 Certified value (μg/g) NIST SRM 1568a Rice flour 0.29 ± 0.03 0.38 ± 0.04 CRM 526 Tuna fish tissue 4.8 ± 0.3 NRC-CNRC DORM-4 Fish protein 6.80 ± 0.64 3.56 ± 0.34 BRC 414 Plankton 6.82 ± 0.28 1.75 ± 0.10 NBS SRM 1646 Estuarine sediment 11.6 ± 1.3 (0.6) a NIST SRM 1566a Oyster tissue 14.0 ± 1.2 2.21 ± 0.24 NRC-CNRC TORT-3 Lobster Hepatopancreas 59.5 ± 3.8 10.9 ± 1.0 Seronorm TM Trace elements Urine, Level 1, Sero, Norway 79 ± 16 b 13.9 ± 2.8 b a Non-certified concentration of constituent element b Unit of reference material, (μg/l), since it is not a solid As Se 3.3 Sample preparation 3.3.1 Digestion procedure Most of the certified reference materials measured during this study are solids, so in order to analyze these, it is necessary to digest the material. This can be accomplished by using an appropriate digestion procedure. A digestion can be performed in both closed and open vessels and using different heat sources, such as microwave and hot plates. In this project, it was chosen to conduct the digestion in a closed vessel due to the fact that As, in the form of AsH 3, is volatile, and to use a hot plate as heat source. The hot plate digestion was preferred over microwave digestion, even though this often results in a longer digestion time. However, the hot plate digestions are easier to perform, less prone to contamination and easier to control. For the development of the digestion procedure, the literature was consulted and it was also taken into consideration that it would not be advantageous to use HCl or HClO 4 as oxidants, since the polyatomic ions 40 Ar 35 Cl + and 40 Ar 37 Cl + interfere with the determination of 75 As and 77 Se, respectively. The final digestion procedure, which was followed, is given below. However, for samples comprising of sedimentary material, this general digestion procedure was found not to be sufficient and to these samples, HF was additionally added in order to also digest the silicates present in the material. For the hot plate digestion, the certified reference materials were accurately weighed in a Savillex PFA vessel. Masses ranging between 0.0800-0.2200 g were used, to which 4 ml of 14 M HNO 3 and 1 ml of 9.8 M H 2 O 2 was added. In case of sediment, an additional 1 ml of 28 M HF Page 22 of 65

was added to the material to fully digest the sample. The mineralization was carried out at 110 C overnight on a hot plate. Additionally, in each set of digestions, blanks were included. After digestion, the digests were quantitatively transferred to centrifuge tube, and centrifuged for 5 min at 7000 rpm and 20 C in order to precipitate any undigested solids and the samples were stored at 5 C until analysis. For the determination of As, a 40-fold dilution with H 2 O was performed to reduce the concentration of the concentrated acids added for the digestion and to reduce the matrix load. For the analysis of Se, however, only a 20-fold dilution with H 2 O was performed, due to the fact that the method for Se determination is less sensitive than the method for As determination and the Se concentration in the reference materials were low. To all samples and standards, Te was added as internal standard, with a final concentration of 5 μg/l and 10 μg/l for the determination of As and Se, respectively. In order to avoid problems with the stability of the solutions and to reduce the risk of contaminating the samples, all samples were measured within 24 h after sample preparation. Additionally, before using of the Savillex PFA vessels, these were cleaned using an extensive procedure listed below in order to avoid possible contamination problems from previous digestions. In step 1, the vessels were rinsed 3 times with H 2 O, whereafter they were left in a soap bath for 24 h. Hereafter, in step 2, the vessels were filled halfway with 7 M HNO 3, which was prepared from 14 M HNO 3 pro-analysis, and left for 24 h on a hotplate at 110 C. Step 3 is the same as step 2, with the addition of new 7 M HNO 3. In Step 4, the vessels were filled with 6 M HCl, which was prepared from 12 M HCl pro-analysis, and left for 24 h on a hotplate at 110 C. Step 5 is the same as step 4, with the addition of new 6 M HCl. In the final step the vessels were left open on a hotplate, and they were removed as soon as they were completely dry. Between each step mentioned above, the vessels were rinsed three times with H 2 O. The procedure was carried out under clean lab conditions (class - 10). 3.3.2 Reconstitution procedure for urine In order to analyze the freeze-dried Seronorm TM reference material, the urine had to be reconstituted prior to analysis. This was done following the procedure provided by the supplier, where firstly the screw cap was removed and the septum was lifted until a groove was present so that air could enter the vial. Secondly, after waiting 5-10 min, the septum was completely removed and 5 ml of H 2 O was added to the vial containing the freeze-dried urine, whereafter the vial was closed completely with the screw cap and left for 30 min. Finally, the material was quantitatively transferred to a Page 23 of 65

Teflon Savillex PFA vessel, in order to prevent contamination from the septum, screw cap and vial, and stored at 5 C until analysis. Prior to analysis, the resulting solution was diluted 20-fold with 0.14 M HNO 3 in order to reduce the matrix load. To all samples and standards, Te was added as internal standard, with a final concentration of 5 μg/l and 10 μg/l for the determination of As and Se, respectively. In order to avoid problems with the stability of the solutions and to reduce the risk of contaminating the samples, all samples were measured within 24 h after sample preparation. Page 24 of 65

4. Results and discussion Through the years, different approaches have been used using Q-ICP-MS equipped with a collision/reaction cell for the determination of As and Se. These approaches often use more conventional gases, such as He, H 2, O 2 and CH 4, since these have proven to be useful, and the prediction of the reaction path of the gases has been more or less straightforward, where e.g., O 2 as a gas in the reaction cell will often result in the atom transfer reaction, M + + ½ O 2 MO +. [32] However for more unconventional gases such as CH 3 F, the reaction gas which will be investigated in this project, five main reaction paths exist, but higher order reaction products can also be formed. Thus, it is difficult to predict the main reaction product. It is first with the introduction of ICP- MS/MS that is has become relatively easy and straightforward to use such a gas, since the product ion scan enables easy determination of the main reaction product(s) formed in the cell. In this work, methods for the determination of As and Se using CH 3 F as a reaction gas have been developed and compared with methods using the more conventional collision gas He and using no gas, by comparing the approaches with regard to analytical performance and the ability to overcome spectral interferences. Likewise, comparisons were also made with SF-ICP-MS. Further validation of the methods using CH 3 F was performed by analyzing a set of various reference materials. 4.1 Optimization of ICP-MS/MS protocol for the determination of As and Se 4.1.1 As For the interference-free determination of As, a method using CH 3 F as reaction gas in MS/MS mode was developed and optimized and likewise, methods using no gas, He in MS/MS mode and CH 3 F in SQ mode were also developed and optimized in order to compare the method using CH 3 F as reaction gas, to these. For all methods developed, the most important optimized settings and parameters are shown in table 2, section 3.1, whereas a full version is given in appendix 1. 4.1.1.1 CH 3 F Starting from a standard method, it was possible to optimize the parameters to fit the problem in question, which was the determination of As free of interference using CH 3 F as reaction gas in MS/MS mode. Firstly it was necessary to identify which product ions were mainly formed. This could be investigated in a fast and easy way by using a product ion scan, where the m/z-ratio of Q1 was set at the original analyte ion mass (75 amu), while Q2 scanned over the entire mass range, thus from 0-260 amu. In order to be able to choose the product ion, which would give the overall Page 25 of 65

highest signal intensity, four product ions scans were conducted using four different cell gas flow rates, 25 %, 50 %, 75 % and 100 %, while measuring a 5 μg/l As standard solution in 0.14 M HNO 3. Figure 16 shows the results of these four scans, with signal intensities as a function of the Q2 m/z-ratio, and the most important reaction products are indicated. The range shown is 0-150 amu, since above this, no more reaction products were present, at the CH 3 F flows used here. Figure 16 Production ion scans for As, Q1: 75 and Q2: scanned, at different cell gas flow rates of CH 3 F as reaction gas for a solution containing 5 μg/l As, where A: 25 %, B: 50 %, C: 75 %, and D: 100 % of the maximum flow rate. Range 10-150 amu. It can be seen from figure 16 that as the cell gas flow rate increases, the spectra become more complex due to the formation of more reaction products, especially in the upper end of the spectrum, which is due to the formation of higher order complexes. In order to make it easier to interpret the information from figure 16, figure 17 only displays the peaks with the highest intensities, which can be used for the determination of As. Page 26 of 65

75 15 75 33 75 49 75 75 75 89 75 94 75 107 75 113 75 123 Intensity (cps) 100000 90000 + AsCH 2 As + 25% 50% 80000 75% 70000 100% 60000 50000 40000 30000 20000 CH 3 + CH 2 F + C 2 H 6 F + AsF + AsCHF + AsF 2 + AsCH 2 (CH 3 F) + 10000 0 Q1 Q2 (amu) Figure 17 Most important conversions in As - CH 3 F reactions at the four different flows investigated: 25, 50, 75 and 100 % of the maximum flow rate. Figure 17 shows the signal intensity at Q2 m/z-ratios of 15, 33, 49, 75, 89, 94, 107, 113 and 123. It can be seen from the figure that at lower m/z-ratios (< 75 amu), charge-transfer product ions are formed such as CH 3 + (m/z-ratio = 15), CH 2 F + (m/z-ratio = 33) and C 2 H 6 F + (m/z-ratio = 49). At an m/z-ratio = 75 unreacted As + is present and it can be seen that as the cell gas flow rate of CH 3 F increases, the signal intensity of As + decreases. This is due to the presence of more gas in the cell, which leads to more collisions and reactions. At higher m/z-ratios (> 75 amu) the product ions, which are formed due to reaction with the analyte ion, can be seen such as AsCH 2 + ( m/z-ratio = 89), which is the result of HF elimination, AsF + (m/z-ratio = 94), which is the result of F atom transfer, AsCHF + (m/z-ratio = 107), which is the result of dehydrogenation, AsF 2 + (m/z-ratio = 113), which also is the result of F atom transfer and AsCH 2 + (CH 3 F) (m/z-ratio = 123), a higher order product, which is the result of both HF elimination and molecular addition. The ion that shows the overall highest signal intensity at a cell gas flow rate of 75 % is AsCH 2 +, and this is thus the main reaction product formed when using CH 3 F. This coincides with literature, where it was likewise found that the main product formed is AsCH 2 + due to HF elimination. [38] Based on this observation, it was chosen to use Q2 at mass 89 during all of the following measurements. Additionally, it can be seen from the figure that the amount of the higher order product AsCH 2 + (CH 3 F) increases as the flow rate goes up and if a higher concentration of CH 3 F in the reaction gas could be used, this might become the main reaction product. Page 27 of 65

Intensity (cps) After having identified the mass that will be selected via Q2, it was now possible to find the optimal flow rate. This was done either manually by observing the signal intensity when changing the cell gas flow rate between 0-100 % or by letting the software run a ramp cell gas scan, where a blank solution, 0.14 M HNO 3 and a 5 μg/l As standard solution were measured at different cell gas flow rates. Figure 18 was obtained by using the setting Q1 = 75 amu Q2 = 89 amu, where signal intensity is shown as a function of cell gas flow rate, using an interval of 5 %. 120000 100000 80000 75 + AsCH 2 5 μg/l As Blank (HNO3, 3 0.14 M) 60000 40000 20000 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Cell gas flow rate (%) Figure 18 Optimization of the CH 3 F reaction gas flow rate. Signal intensities are shown as a function of the cell gas flow rate for a 0.14 M HNO 3 blank solution (blue) and a 5 μg/l As standard solution (red). It can be seen from figure 18 that the highest signal intensity is obtained for a cell gas flow rate of 70 %, thus the optimal cell gas flow is around a flow of 70 %. The final fine-tuning of the optimal cell gas flow could be done manually, as described earlier, and it was found that at a flow of 72 % the signal intensity was the highest and this was set as the optimal cell gas flow. Hereafter, it was possible to fine-tune all the other parameters of the method in order to obtain the highest sensitivity. This was done either manually and/or by using auto tune, where the software performs the optimization of some of the parameters. This method is further on denoted as MS/MS-CH 3 F-As. The method developed above was for MS/MS mode, using CH 3 F as reaction gas. Similarly it is possible to develop a method for SQ mode, using CH 3 F as reaction gas. The only difference between the two methods is the fact that in the SQ mode, Q1 is operated in "fully open" mass width. Further on this method is denoted as SQ-CH 3 F-As. Page 28 of 65

4.1.1.2 No gas In order to assess the advantages of the MS/MS-CH 3 F-As method, a method was developed to determine As using no gas in the cell in MS/MS mode. It is however expected that a method using no gas will not be able to deal well with spectral overlap, due to the fact that no mechanism is present to remove the on-mass interferences from, e.g., 40 Ar 35 Cl +. A method was developed by again starting from a standard method and optimizing the parameters in order to determine As using no cell gas. Due to no gas being present in the collision/reaction cell, there was no need for the identification of the main product ion, since the analyte ion to be measured will be 75 As +, thus Q1 and Q2 were set at mass 75. Knowing this, it was possible to fine-tune all the parameters of the method in order to obtain the highest signal intensity and thus the highest sensitivity. This could be done either manually and/or by using auto tune, similar to the way the parameters were optimized for the MS/MS-CH 3 F-As method. This method is further on denoted as MS/MS-no gas-as. 4.1.1.3 He Furthermore, a method using He as collision gas in MS/MS mode was also developed and optimized to evaluate the advantages of the MS/MS-CH 3 F-As method. The interference-free determination of As using He as collision gas is based on the idea that it is possible to remove the major on-mass polyatomic interference of 40 Ar 35 Cl +, owing to the fact that polyatomic ions are larger and thus will collide more frequently with the He atoms and thus, lose their charge, dissociate and/or lose a substantial amount of kinetic energy, enabling to discriminate against these ions. [39,49] Starting from a standard method, it was possible to optimize the parameters in order to determine As free of interference using He as collision gas. No product ion scan had to be performed, due to the fact that He is a collision gas, thus the analyte ion will not engage in reaction forming a product ion. However the cell gas flow of He had to be optimized, in order to find the flow, at which signal intensity and thus, the sensitivity was highest, but also where the flow was high enough to eliminate the polyatomic interference from 40 Ar 35 Cl +. This was investigated by measuring two standard solutions, a 5 μg/l As standard solution in 0.14 M HNO 3 and a 5 μg/l As standard solution with 1000 mg/l Cl in 0.14 M HNO 3 interchangeably, at increasing flow rates of He and with Q1 and Q2 set at mass 75. The result can be seen in figure 19, where the signal intensity is shown as a function of cell gas flow rate. Page 29 of 65

Intensity (cps) 80000 70000 60000 5 μg/l As 5 μg/l As + 1000 mg/l Cl 50000 40000 30000 20000 10000 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Cell gas flow rate (ml/min) Figure 19 Optimization of the He collision gas flow rate. Signal intensities are shown as a function of the cell gas flow rate for a 5 μg/l As standard solution (blue) and a 5 μg/l As standard solution with 1000 mg/l Cl (red). From the figure, it can be seen that at a flow of 1.5-2.0 ml He/min the highest signal intensity is obtained and at a flow of 3 ml He/min, the two lines coincide, indicating that a minimum flow of 3 ml He/min is needed in order to remove the polyatomic interference from 40 Ar 35 Cl +. As one goes to higher flow rates, the signal intensity decreases, thus it would be natural to choose a flow of 3 ml He/min as the optimal flow. However, the recommendations from the company for the MS/MS mode using He as collision gas, states that it is recommended to use a flow of 4-5 ml He/min. Thus it was chosen to use a cell gas flow of 4 ml He/min even though this meant that a loss in signal intensity would be obtained in comparison to using 3 ml He/min. After finding optimal flow rate, all other parameters of the method could be fine-tuned to obtain the highest sensitivity. This was done in a similar way to what was described for the MS/MS-CH 3 F-As method. This method is further on denoted as MS/MS-He-As. 4.1.2 Se As for As, a method to determine Se interference-free using CH 3 F as reaction gas in MS/MS was developed and optimized. In this project the Se - isotopes 77 Se, 78 Se and 80 Se were monitored, since these isotopes are typically monitored in quadrupole ICP-MS, with 80 Se and 78 Se being the two most abundant isotopes. Additionally a method, where more than one isotope could be determined, was sought, since this opens up possibilities for isotopic analysis. Page 30 of 65

Likewise a method in SQ mode using CH 3 F as reaction gas and a method using He as collision gas in MS/MS mode were also developed and optimized in order to compare these with the method using CH 3 F as reaction gas in the MS/MS mode. However no method using no gas was developed for Se in order to protect the detector, due to the presence of 40 Ar 40 Ar + and 40 Ar 38 Ar + in large amounts, which interfere with the analyte ions 80 Se + and 78 Se +, respectively, and in the no gas method no mechanism is present that can reduce these interferences. For all methods developed, the most important optimized settings and parameters are shown in table 2, section 3.1, whereas a full version is given in appendix 1. 4.1.2.1 CH 3 F As was the case for As, the starting point is again a standard method, that is optimized to fit the problem in question, which is the interference-free determination of Se using reaction gas CH 3 F in MS/MS mode. It was chosen to perform the development and optimization of the method for 80 Se, since it has the highest abundance of the Se - isotopes and it is also the most interfered isotope. As for the method developed for As, it was firstly necessary to identify which product ions were primarily formed. This was again done by performing a product ion scan, where the m/z-ratio of Q1 was set at a fixed value - the m/z-ratio of the original analyte ion (80 amu) - while Q2 scanned over the entire mass range, thus from 0-260 amu. In order to choose the product ion with the overall highest signal intensity, four product ions scans were conducted at the same flows of CH 3 F as used for the method development for As and measuring a 5 μg/l Se standard solution in 0.14 M HNO 3. Figure 20 shows the results of these four scans, with signal intensities as a function of the Q2 m/zratio, and the most important reaction products are indicated. In the figure, only the range 10-120 amu is shown, because no reaction products are present above this m/z-ratio. Page 31 of 65

Figure 20 Production ion scans for Se, Q1: 80 and Q2: scanned, at different cell gas flow rates of CH 3 F as reaction gas for a solution containing 5 μg/l Se, where A: 25 %, B: 50 %, C: 75 %, and D: 100 % of the maximum flow rate. Range 10-120 amu. It can be seen from figure 20 that as the cell gas flow rate increases, the spectra do not become more complex as was seen for As, where more complex species could be seen at higher flow rates. This is due to the absence of higher order complex formation for Se at the investigated CH 3 F flow rates. In order to make it easier to interpret the information that can be obtained from figure 20, the peaks, with the highest intensities that could be used for Se determination, are displayed in figure 21. Page 32 of 65

80 15 80 33 80 49 80 80 80 94 Intensity (cps) 100000000 10000000 1000000 CH 3 + CH 2 F + C 2 H 6 F + Se + 25% 50% 75% 100% 100000 10000 SeCH 2 + 1000 100 10 1 Q1 Q2 (amu) Figure 21 Most important conversions in Se - CH 3 F reactions at the four different flows investigated: 25, 50, 75 and 100 % of the maximum flow rate. Figure 21 shows the signal intensity at Q2 m/z-ratios of 15, 33, 49, 80, and 94. From the figure, it can be seen that at lower m/z-ratios (< 80 amu) charge-transfer product ions are formed, such as CH 3 + (m/z-ratio = 15), CH 2 F + (m/z-ratio = 33) and C 2 H 6 F + (m/z-ratio = 49). These product ions were likewise observed for As. At an m/z-ratio = 80 unreacted Se + is present and it can be seen that as the cell gas flow rate of CH 3 F increases, the signal intensity of Se + decreases due to more collisions and reactions taking place. This trend was also observed for unreacted As. At higher m/zratios (> 80 amu) product ions, which are formed due to reaction with the analyte ion, can be seen, such as SeCH 2 + (m/z-ratio = 94), which is the result of HF elimination. The ion which shows the overall highest signal intensity is CH 2 F +, however this ion is not appropriate to use for the determination of Se, due to the fact that the amount of the product ion CH 2 F + is not proportional to the amount of Se present, since charge transfers can occur from all ions present in the reaction cell and not just from 80 Se +. The highest signal intensity, for ions which are not produced due to charge transfer, is from the ion of the unreacted 80 Se +, but this ion is also not appropriate to use for the determination of Se due to the fact that it suffers from interference from, e.g., 40 Ar 40 Ar +. Thus, the only suitable ion to use is the reaction product ion SeCH 2 +, and it can be seen that the signal intensities of this ion increase as the flow goes up with a maximum signal intensity at a flow of 100 %. This type of reaction product ion - MCH 2 + - was also observed for the reaction of As with CH 3 F, which makes sense since As and Se are located next to each other in the periodic table and thus, it Page 33 of 65

Intensity (cps) would be highly likely that these two would react in a similar way with CH 3 F. However, in literature, it can be found that, as mentioned before, As is expected to form the reaction product AsCH + 2 upon reaction with CH 3 F in a reaction cell, whereas for Se the same paper states that no reaction takes place between Se and CH 3 F. [38] This difference may be due to the use of a different type of instrumentation and settings than that of the paper. However, in this work, SeCH + 2 was observed as a reaction product, and it was decided to use Q2 at a mass of 94 during all of the following measurements of 80 Se. It is now possible to find the optimal cell gas flow rate, by either observing the signal intensity while manually changing the cell gas flow rate between 0-100 % or, as was also done for As, the software could perform a ramp cell gas scan, where a blank solution, 0.14 M HNO 3 and a 5 μg/l Se standard solution were measured at different cell gas flow rates. Figure 22 shows the result of the ramp cell gas scan, where signal intensity is shown as a function of cell gas flow rate, using an interval of 5 %. 12000 10000 8000 6000 80 SeCH 2 + 5 μg/l Se Blank (HNO3, 3 0.14 M) 4000 2000 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Cell gas flow rate (%) Figure 22 Optimization of the CH 3 F reaction gas flow rate. Signal intensities are shown as a function of the cell gas flow rate for a 0.14 M HNO 3 blank solution (blue) and a 5 μg/l Se standard solution (red). From figure 22, it can be seen that the highest signal intensity is obtained for a cell gas flow rate of 100 %, and this was set as the optimal cell gas flow. However, from looking at the curve of the 5 μg/l Se standard solution, it can be seen that at a flow of 100 %, the signal is at its maximum, which may indicate that if a higher cell gas flow was possible by, e.g., using another CH 3 F/He mixture or by attaching it to the 3 rd gas inlet, where higher flow rates are possible, a higher signal intensity and thus, a higher sensitivity might be obtained. However, due to the gas being attached to Page 34 of 65

the 4 th gas inlet, the maximum flow rate possible was 100 % (as mentioned in the experimental section). Knowing the optimal cell gas flow, the other parameters of the method could be optimized in order to obtain the highest sensitivity. This was done similar to the method for As, by observing when a signal intensity increase was observed upon manually changing the settings and/or by using auto tune. This method is further on denoted as MS/MS-CH 3 F-Se. Furthermore, a method for SQ mode using CH 3 F as reaction gas was also developed in a similar way as for As, where the SQ mode is similar to the MS/MS mode with the only difference being that in SQ mode, Q1 operates in "fully open" mass width. This method is further on denoted as SQ- CH 3 F-Se. 4.1.2.2 He To assess the advantages of the MS/MS-CH 3 F-Se method, a method using He as collision gas in MS/MS mode was developed and optimized. As mentioned earlier, the three Se - isotopes 77 Se, 78 Se and 80 Se, will be monitored, and for the development of the MS/MS-CH 3 F-Se method, 80 Se, the isotope with the highest abundance, was used. However, for the development and optimization of this method, it was chosen to use the isotope 77 Se instead, since it is the only isotope that does not suffer from ArAr + interference ( 40 Ar 40 Ar + and 40 Ar 38 Ar + ). Likewise, starting from a standard method, it is possible to optimize the method to enable determination of Se using He as collision gas. The idea behind the use of He as collision gas is similar to that described for As, and in order to optimize the method, no product ion scan had to be performed, since He is only a collision gas. However, the flow rate of He had to be optimized, in order to obtain the highest signal intensity and thus, the highest sensitivity, as well as being able to eliminate polyatomic interferences effectively. This was done by measuring a 5 μg/l Se standard solution and a blank solution, 0.14 M HNO 3 interchangeably at increasing flows of He to observe when the major interference of 40 Ar 38 Ar + on 78 Se + was removed. In figure 23, the results are displayed, where signal intensity is shown as a function of cell gas flow rate. Page 35 of 65

Intensity (cps) 40000 35000 30000 5 μg/l Se Blank (HNO3, 3 0.14 M) 25000 20000 15000 10000 5000 0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Cell flow gas rate (ml/min) Figure 23 Optimization of He collision gas flow rate. Signal intensities are shown as a function of the cell gas flow rate for a 5 μg/l Se standard solution (red) and a 0.14 M HNO 3 blank solution (blue). It can be seen from figure 23 that, as the flow rate of He increases, the signal intensity of the blank decreases, indicating that the interference from ArAr + is reduced, but a decrease is also seen for the Se standard, which is due to both a reduction of the ArAr + interference and a reduction of the transmission of the analyte ion Se +. At a flow of 4 ml He/min, the interference from 40 Ar 38 Ar + is removed, since the intensity of the blank signal is close to zero, and this was chosen as the optimal He flow rate. The method was additionally optimized by fine-tuning all the other parameters of the method, either manually and/or by using auto tune, similar to what was done for the Se using CH 3 F as reaction gas. Further on this method is denoted as MS/MS-He-Se. However, this method was only used to measure the isotopes 77 Se and 78 Se, since 80 Se suffers from a very pronounced interference from 40 Ar 40 Ar + and in order to protect the detector, it was chosen not to investigate if it would be possible to monitor 80 Se using He as collision gas. 4.2 Optimization of SF-ICP-MS protocol for the determination of As and Se In order to further evaluate the MS/MS-CH 3 F-As and MS/MS-CH 3 F-Se methods, a method using a different technique, SF-ICP-MS, was developed, since this is the typical technique used for interference-free determination of As and Se nowadays. The polyatomic interferences from, e.g., 40 Ar 35 Cl + on 75 As +, 40 Ar 37 Cl + on 77 Se + and 40 Ar 38 Ar + on 78 Se + could be resolved using high resolution. However the polyatomic interference from 40 Ar 40 Ar + on 80 Se + could not be overcome in high resolution, since this requires a theoretical resolution of 9688 [26], which is very close to the Page 36 of 65

maximum resolution attainable with the SF-instrument, especially when taking into account that the intensity of the 40 Ar 40 Ar + peak is a lot higher than that of the 80 Se + peak. Thus only 75 As and the two Se - isotopes 77 Se and 78 Se were monitored using SF-ICP-MS. The most important optimized parameters and settings are shown in table 3, section 3.1. 4.3 Calibration data and limits of detection 4.3.1 As The analytical performance of the method for interference-free determination of As using CH 3 F was compared to the analytical performance of all the other methods developed. The sensitivity and instrumental limits of detection and quantification of the methods developed along with other parameters were assessed by measuring standard solutions of 0, 0.5, 1, 2.5 and 5 μg/l As in 0.14 M HNO 3. 10 consecutive replicate measurements were performed to determine the standard deviation of the blank, the slope and the intercept. From the measurements, it was possible to calculate the limit of detection (LOD) and limit of quantification (LOQ) as 3 and 10 times the standard deviation on the blank divided by the slope, respectively. [47] In table 5, the results are presented for the various ICP-QQQ methods, as well as for the Element XR SF-ICP-MS. The calibration curves can be seen in appendix 2. Table 5 Calibration data and instrumental LODs and LOQs obtained for As with ICP-MS/MS (several modes) and SF-ICP- MS. Set-up mode Reaction gas Q1 (amu) Q2 (amu) Sensitivity a (L/μg) Intercept a (count s -1 ) R 2 LOD b (μg/l) LOQ b (μg/l) MS/MS CH 3 F 75 89 18160 ± 250-90 ± 100 0.999994 0.0005 0.002 MS/MS No gas 75 75 28120 ± 73 133 ± 80 0.999992 0.0009 0.003 MS/MS He 75 75 3914 ± 40-11 ± 37 0.9999989 0.002 0.006 SQ CH 3 F - 89 34710 ± 580-20 ± 170 0.999987 0.001 0.004 Nuclide monitored SF-ICP-MS 75 As 824 ± 18 13 ± 12 0.99996 0.01 0.04 a Uncertainties are expressed as standard deviation (n=10) b LOD and LOQ are calculated as 3 and 10 times the standard deviation of a blank solution (0.14 M HNO 3 ) divided by the slope of the calibration curve, respectively, using 10 consecutive replicate measurements of the blank to obtain the standard deviation. From table 5, it can be seen that the highest sensitivity is obtained with the SQ-CH3F-As method, the second highest with MS/MS-no gas-as, followed by MS/MS-CH 3 F-As, while MS/MS-He-As is the method, which shows the lowest sensitivity of the methods using the Agilent 8800 ICP-QQQ. This complies with what is to be expected, since in SQ mode Q1 operates in fully open mode and Page 37 of 65

As + is more efficiently transported. Additionally, when comparing the sensitivities obtained in the MS/MS mode, the MS/MS-no gas-as method has the highest sensitivity, which is also what would be expected, since no gas is present in the cell with which As can collide or react with, losing its positive charge or forming a reaction product. However, the sensitivity obtained with the MS/MS- CH 3 F-As method is comparable to that obtained using no gas, indicating that the efficiency of the reaction is high. Additionally, it can be seen that the lowest overall sensitivity was obtained using the Element XR SF-ICP-MS, which is due to the use of the highest resolution setting, which significantly reduces the sensitivity, but high resolution is necessary in order to resolve the interference from, e.g., 40 Ar 35 Cl, which requires a theoretical resolution of 7773. [26] For all calibration curves, a value of > 0.9999 was obtained for R 2, which shows that the response is proportional to the amount of analyte. When comparing the instrumental LODs, it can be seen that the lowest LOD is obtained for the MS/MS-CH 3 F-As method with 0.0005 μg/l, followed by MS/MS-no gas-as, SQ-CH 3 F-As and MS/MS-He-As. However, it should be noted that these LODs do not give a good indication about the real strength of the methods for dealing with spectral interferences, as they have been calculated on the basis of measurements of pure standards and blanks. Therefore, the low LODs found for the MS/MS-no gas-as and MS/MS-He-As methods can be misleading. In the MS/MS-no gas-as method, no gas is present in the collision/reaction cell, which means that this method is not able to cope with interferences, such as e.g., 40 Ar 35 Cl +, which may be present when measuring a real sample, and with the MS/MS-He-As method it is not possible to remove doubly charged interfering ions, as will be demonstrated later on. The method with the highest LOD, is the Element XR SF- ICP-MS. In comparison to values that have previously been reported when measuring As, the instrumental detection limit obtained with MS/MS-CH 3 F-As is definitely better than those obtained using quadrupole ICP-MS (1.35 μg/l), where correction equations have also been used [19], doublefocusing sector field ICP-MS (0.004 μg /L) [33] and quadrupole-based ICP-MS equipped with a collision/reaction cell using gases, such as H 2 /He (0.15 μg/l) [19] or other techniques such as atomic absorption spectrometry (0.3 μg/l) [50]. Thus, this shows that with the MS/MS-CH 3 F-As method, it is possible to obtain an instrumental LOD that is the lowest of what could be measured, but also of what could be found in literature. Page 38 of 65

4.3.2 Se Also for Se, the analytical performance of the method using CH 3 F as reaction gas was compared to that of all the other methods developed. Again sensitivity, instrumental limits of detection and quantification and other parameters were assessed by measuring 10 consecutive replicate measurements of standard solutions of 0, 0.5, 1, 2.5 and 5 μg/l Se in 0.14 M HNO 3. Table 6 shows the results obtained for the various ICP-QQQ methods, as well as for the Element XR SF-ICP-MS. As for As, the calibration curves can be seen in appendix 2. Table 6 Calibration data and instrumental LODs and LOQs obtained for Se with ICP-MS/MS (several modes) and SF-ICP- MS. Set-up mode Reaction gas Q1 (amu) Q2 (amu) Sensitivity a (L/μg) Intercept a (count s -1 ) R 2 LOD b (μg/l) MS/MS CH 3 F 77 91 286 ± 10 3 ± 11 0.99998 0.02 0.07 MS/MS CH 3 F 78 92 917 ± 20 7 ± 13 0.999986 0.009 0.03 MS/MS CH 3 F 80 94 1944 ± 25-2 ± 22 0.99997 0.007 0.02 MS/MS He 77 77 302 ± 8 0 ±13 0.99995 0.04 0.1 MS/MS He 78 78 997 ± 22 99 ± 23 0.999991 0.06 0.2 SQ CH 3 F - 91 728 ± 26 3146 ± 72 0.9996 0.2 0.8 SQ CH 3 F - 92 1945 ± 20 317 ± 43 0.9997 0.02 0.06 SQ CH 3 F - 94 4075 ± 30 175 ± 33 0.99997 0.01 0.04 Nuclides monitored LOQ b (μg/l) SF-ICP-MS 77 Se 67 ± 5 2 ± 5 0.9999 0.05 0.2 SF-ICP-MS 78 Se 223 ± 8 28 ± 17 0.9993 0.5 2 a Uncertainties are expressed as standard deviation (n=10) b LOD and LOQ are calculated as 3 and 10 times the standard deviation of a blank solution (0.14 M HNO 3 ) divided by the slope of the calibration curve, respectively, using 10 consecutive replicate measurements of the blank to obtain the standard deviation. It can be seen from table 6 that the highest sensitivity was obtained for the SQ-CH 3 F-Se method followed by MS/MS-CH 3 F-Se, with which comparable sensitivities are obtained as with MS/MS- He-Se. As for As, the method with the lowest sensitivity is the Element XR SF-ICP-MS, which again is due to the use of the highest resolution setting, which results in a reduction of sensitivity in the order of 2 magnitudes, but again high resolution is necessary for the determination of Se, since the interference from, e.g., 40 Ar 37 Cl + on 77 Se + requires a theoretical resolution of 9182 and 40 Ar 38 Ar + on 78 Se + requires a theoretical resolution of 9970. [26] Furthermore, it can be seen that the sensitivity obtained for the MS/MS-CH 3 F-Se method is not very high in comparison to what was found for As, since Se has multiple isotopes and the reaction to form SeCH 2 + is less favorable. For all calibration curves, a value above 0.999 was obtained for R 2. Page 39 of 65

From looking at the instrumental LODs that were obtained, it can be seen that the MS/MS-CH 3 F-Se method has the lowest LOD of 0.007 μg/l via 80 Se, followed by 78 Se. This is then followed by MS/MS-CH 3 F-Se via ( 78 Se), SQ-CH 3 F-Se via ( 78 Se and 80 Se), MS/MS-CH 3 F-Se via ( 77 Se), MS/MS-He-Se and SF-ICP-MS via ( 77 Se), which all have comparable LODs. The highest LODs are found for SQ-CH 3 F-Se via ( 77 Se) and SF-ICP-MS via ( 78 Se). However as for As, the LODs do not give a good indication about the real strength of the methods for dealing with interferences, as the method using He as collision gas, e.g., is unable to overcome doubly charged interferences, since this method has no mechanism to remove these, as will be demonstrated later on. When comparing the values obtained in this project with values reported in literature, it can be found that the instrumental LOD obtained for MS/MS-CH 3 F-Se via 80 Se is definitely better than those obtained using Q-ICP-MS (0.28 μg/l via 82 Se + and by using correction equations) [19] and Q-ICP-MS equipped with a collision/reaction cell using other gases than CH 3 F such as H 2 /He (0.029 μg/l via 80 Se + ) [19], and comparable to those obtained using SF-ICP-MS (0.004 μg /L via 82 Se + ) [34]. Thus even though the reaction of Se with CH 3 F is less favorable than that of As, low LODs are still found. 4.4 Investigation of the improvement in sensitivity by the addition of MeOH 4.4.1 As As mentioned in the introduction, As and Se have high ionization potentials and thus are poorly ionized and in literature, it can be found that carbon, e.g., in the form of MeOH [4,29,31], can be added to induce the carbon effect and thereby increase the sensitivity for the analyte. With the increase in sensitivity, it might also be possible to decrease the limit of detection and in ultra-trace level determinations, it is the aim to be able to determine the analyte at as low concentrations as possible. However, it also has to be mentioned that the addition of carbon can also be used to resolve the problem of the carbon effect when standards and samples differ in matrix if it is not possible to find an appropriate internal standard that experiences a similar enhancement as the analyte. In order to investigate the increase in sensitivity for As, solutions containing 5 μg/l As standard solution in HNO 3 /MeOH - 100/0 %, 98/2 %, 96/4%, 94/6%, 92/8%, 90/10 % and the corresponding blank solutions were prepared and analyzed using the MS/MS-CH 3 F-As method. Figure 24 shows the results, both as signal intensity and as ratio, calculated as signal 5 μg/l As / signal blank, as a function of MeOH concentration. Page 40 of 65

Intensity (cps) Ratio 250000 200000 150000 100000 50000 0 5 μg/l As Blank (HNO3, 3, 0.14 M) Ratio 0 2 4 6 8 10 Conc. MeOH (%) 3000 2500 2000 1500 1000 500 0 Figure 24 Investigation of the influence of the addition of MeOH. Signal intensity is shown as a function of MeOH concentration for a blank solution, 0.14 M HNO 3 with increasing MeOH concentration (blue), and a 5 μg/l As standard solution 0.14 M HNO 3 with increasing MeOH concentration (red). Additionally, the ratio is also shown as a function of MeOH concentration, where the ratio (green) is calculated as signal 5 μg/l As / signal blank. It can be seen from figure 24 that with 94/6 % HNO 3 /MeOH, the highest signal intensity is seen for the 5 μg/l As standard solution and the signal intensity has doubled in comparison to no addition of MeOH. However as the amount of MeOH goes up, the intensity for the blank also increases due to the impurities in the MeOH and due to an increase in the sensitivity for the blank. When calculating the ratio between the signal for the 5 μg/l As and the blank solution, a decreasing relationship is observed (green ratio curve). Thus, even though the signal intensity for 5 μg/l As standard solution is more than doubled when using 94/6 % HNO 3 /MeOH, the signal for the blank has increases even more. Additionally, above 6 % MeOH, a signal decrease is observed for the 5 μg/l As standard solution, which can be linked to a decrease in plasma efficiency, since the plasma was cooled down by the introduction of MeOH. In order to determine if addition of MeOH would be beneficial, the analytical performance was assessed using a calibration curve prepared using 94/6 % HNO 3 /MeOH in the concentration range 0-5 μg/l of a As standard solution. The results are given in table 7, together with the results obtained for a calibration curve prepared in pure HNO 3. The calibration curve can be seen in appendix 3. Page 41 of 65

Intensity (cps) Ratio Table 7 Calibration data and instrumental LODs and LOQs obtained for As with ICP-MS/MS using MS/MS-CH 3 F-As and HNO 3 and HNO 3 /MeOH 94/6 % as solvent. Set-up mode Reaction gas Q1 (amu) Q2 (amu) Solvent Sensitivity a (L/μg) Intercept a (count s -1 ) R 2 LOD b (μg/l) MS/MS CH 3 F 75 89 HNO 3 18160 ± 250-90 ± 100 0.999994 0.0005 0.002 MS/MS CH 3 F 75 89 94/6 % HNO 3 /MeOH 45700 ± 1200-700 ± 1700 0.9999 0.001 0.004 a Uncertainties are expressed as standard deviations (n=10) b LOD and LOQ are calculated as 3 and 10 times the standard deviation of a blank solution (0.14 M HNO 3 ) divided by the slope of the calibration curve, respectively, using 10 consecutive replicate measurements of the blank to obtain the standard deviation. From table 7, it can be seen, that when using 94/6 % HNO 3 /MeOH as solvent, a sensitivity increase is indeed observed, which coincides with what was seen in figure 24. However, when looking at the instrumental LODs, it is seen that the LOD, for the method using 94/6 % HNO 3 /MeOH as solvent, is higher than that with the method using no MeOH. Thus, it was decided not to use the addition of MeOH further on in this project, since this study is aimed at determining As at ultra-trace levels and for this purpose, an as low as possible LOD is preferred. LOQ b (μg/l) 4.4.2 Se Also for Se, it was investigated if it would be beneficial to add MeOH to the solvent by measuring solutions containing 5 μg/l Se and blank solutions in HNO 3 with 0-10 % MeOH using the MS/MS-CH 3 F-Se method. Figure 25 shows the results, with both signal intensity and ratio, which is calculated as signal 5 μg/l Se / signal blank, as a function of the MeOH concentration. 30000 25000 20000 15000 10000 5000 0 5 μg/l Se Blank (HNO3, 3, 0.14 M) Ratio 0 2 4 6 8 10 Conc. MeOH (%) 200 180 160 140 120 100 80 60 40 20 0 Figure 25 Investigation of the influence of the addition of MeOH. Signal intensity is shown as a function of MeOH concentration for a blank solution, 0.14 M HNO 3 with increasing MeOH concentration (blue), and a 5 μg/l Se standard solution 0.14 M HNO 3 with increasing MeOH concentration (red). Additionally, the ratio is also shown as a function of MeOH concentration, where the ratio (green) is calculated as signal 5 μg/l Se / signal blank. Page 42 of 65

From figure 25, it is seen that with 94/6 % HNO 3 /MeOH, the highest signal intensity is observed for the 5 μg/l Se standard solution, with the signal intensity doubled in comparison to no addition of MeOH. However, also here, the signal intensity of the blank increases with increasing MeOH concentration, which again is due to the impurities present in the MeOH used and due to a sensitivity increase for the blank as well. A decreasing relationship is observed (green ratio curve), when the ratio between the signal for the 5 μg/l Se and the blank solution is calculated. Thus, the signal for the blank has increased even more than the signal for 5 μg/l Se standard solution. As for As, above 6 % MeOH, a signal decrease is observed for the 5 μg/l Se standard solution. Similar to what was done for As, a calibration curve in the concentration range 0-5 μg/l Se standard solution in 94/6 % HNO 3 /MeOH was measured to assess if the addition of MeOH would be favorable. In table 8, the calibration data are given together with the results obtained for a calibration curve prepared in pure HNO 3 and the calibration curve can be seen in appendix 3. Table 8 Calibration data and instrumental LODs and LOQs obtained for Se with ICP-MS/MS using MS/MS-CH 3 F-Se, and HNO 3 and HNO 3 /MeOH 94/6 % as solvent. Set-up mode Reaction gas Q1 (amu) Q2 (amu) Solvent Sensitivity a (L/μg) Intercept a (count s -1 ) R 2 LOD b (μg/l) MS/MS CH 3 F 77 91 HNO 3 286 ± 10 3 ± 11 0.99998 0.02 0.07 MS/MS CH 3 F 78 92 HNO 3 917 ± 20 7 ± 13 0.999986 0.009 0.03 MS/MS CH 3 F 80 94 HNO 3 1944 ± 25-2 ± 22 0.99997 0.007 0.02 MS/MS CH 3 F 77 91 94/6 % HNO 3 /MeOH 725 ± 12 38 ± 21 0.9999 0.02 0.07 MS/MS CH 3 F 78 92 94/6 % HNO 3 /MeOH 2295 ± 41 128 ± 29 0.9999 0.02 0.06 LOQ b (μg/l) MS/MS CH 3 F 80 94 94/6 % HNO 3 /MeOH 5042 ± 80 263 ± 42 0.9999 0.009 0.03 a Uncertainties are expressed as standard deviations (n=10) b LOD and LOQ are calculated as 3 and 10 times the standard deviation of a blank solution (0.14 M HNO 3 ) divided by the slope of the calibration curve, respectively, using 10 consecutive replicate measurements of the blank to obtain the standard deviation. It can be seen in table 8, that a sensitivity increase is observed for all isotopes when using 94/6 % HNO 3 /MeOH as solvent, as expected from the results in figure 25. When looking at the limits of detection obtained, it can however be seen that the LODs for the method using 94/6 % HNO 3 /MeOH as solvent are comparable or slightly higher than those without MeOH. Thus, the overall LOD is not better than what is obtained via the isotope 80 Se using no MeOH and additionally, by adding MeOH the sample preparation also gets more complex. Thus, it was decided, as for As, not to use the addition of MeOH further on in this project when measuring Se. Page 43 of 65

4.5 Results obtained for simulated matrices 4.5.1 As To further validate the method for determination of As using CH 3 F as reaction gas in MS/MS mode, different simulated matrices were prepared and measured in order to demonstrate that with this method, it is possible to remove interferences that often cause problems in the determination of As using other methods, such as no gas and He in MS/MS mode and CH 3 F in SQ mode. This was done by investigating the most important interferences that are present at mass 75, and thus could interfere with the measurement of the analyte ion 75 As + and by investigating potential interferences introduced, when a reaction product is measured instead of the original analyte ion, when using CH 3 F as reaction gas. Table 9 lists the interferences investigated using simulated matrices. Table 9 List of on-mass and mass-shift interferences investigated using simulated matrices for As. Type of interference Mass (amu) Interference On-mass interference of 75 As + 75 40 Ar 35 Cl +, 150 Nd 2+, 150 Sm 2+ Mass-shift interference due to the use of CH 3 F as reaction gas forming the reaction product AsCH 2 + 89 89 Y +, 70 Ge 19 F +, 70 Zn 19 F + Simulated matrices containing 5 μg/l As standard solution with 500 mg/l Cl and with 100 μg/l Nd and Sm were investigated in the MS/MS mode, using the gases CH 3 F and He, and using no gas. Additionally, in the MS/MS mode and in the SQ mode using CH 3 F as reaction gas, the following simulated matrices were investigated: 5 μg/l As standard solution with 1 μg/l Y, with 5 μg/l Ge and with 10 μg/l Zn. The concentrations of the elements investigated here were chosen in such a way that the concentration was high enough to observe the interference, but not more than this, in order to minimize contamination of the system and to protect the detector. For comparison, a pure 5 μg/l As standard solution (no matrix elements added) was also measured in all modes. All samples contained 10 μg/l Se, which was used as internal standard (using the isotope 78 Se + ). When these experiments were performed, in the early stages of the project, Se was selected as internal standard, since it has a similar mass and ionization potential as As, and it was added to be able to correct for signal suppression and/or enhancement. However, in experiments later on, Se has been replaced by Te as internal standard, since Se was present in some of the reference materials investigated. Figure 26 shows the results obtained when measuring the simulated matrices in the different modes with the different gases and by using external calibration. The red line indicates the concentration of 5 μg/l As, as present in all solutions. Page 44 of 65

[As] (μg/l) 15.0 12.5 10.0 (3) (4) (5) MS/MS, CH3F 3 F MS/MS, no gas MS/MS, He SQ, CH3F 3 F 7.5 (1) (2) (6) 5.0 2.5 0.0 5 μg/l As 5 μg/l As + 500 mg/l Cl 5 μg/l As + 100 μg/l Nd/Sm 5 μg/l As + 1 μg/l Y 5 μg/l As + 5 μg/l Ge 5 μg/l As + 10 μg/l Zn Figure 26 Simulated matrices. Concentration of As measured with MS/MS-CH 3 F-As (blue), with MS/MS-no gas-as (red), with MS/MS-He-As (green) and with SQ-CH 3 F-As (purple) is plotted against the different simulated matrices. (n = 5) It can be seen from figure 26 that in all modes, it was possible to obtain 5 μg/l As when no matrix elements were present (1). However, in the case of the simulated matrix containing 5 μg/l As and 500 mg/l Cl (2), more than 5 μg/l As was found with the MS/MS-no gas-as method, which is due to the method not being able to overcome the polyatomic interference from 40 Ar 35 Cl +, which is also present at mass 75. The other methods were able to overcome this interference and accurate results were obtained. With the MS/MS-He-As method the interference was overcome due to collision of the polyatomic interfering ion with the He atoms and subsequent kinetic energy discrimination, while with the MS/MS-CH 3 F-As method, the interference was overcome by using the mass-shift method, (Q2 = 89), where As was measured at mass 89 as AsCH 2 +. In the case where the simulated matrix contains 5 μg/l As and 100 μg/l Nd and Sm (3), it was not possible to obtain accurate results with both MS/MS-no gas-as and MS/MS-He-As. This is due to both methods not being able to remove the doubly charged 150 Nd 2+ and 150 Sm 2+ ions, which appear at the m/z-ratio 75. However, with the MS/MS-CH 3 F-As method accurate results were obtained by again overcoming the interferences due to the mass-shift method (Q2 = 89), where As is measured at mass 89 as AsCH 2 +, where there is no interference present from the doubly charged ions. For the simulated matrices, which contain 5 μg/l As and 1 μg/l Y (4) and 5 μg/l Ge (5), respectively, it can be seen that with the SQ-CH 3 F-As method more than 5 μg/l As was obtained. This is due to the method not being able to remove the interference from 89 Y + and from 70 Ge 19 F +, Page 45 of 65

which was formed in the reaction cell by F atom transfer from the reaction gas CH 3 F, since these ions overlap with the mass of the reaction product ion formed in the reaction cell, AsCH + 2 (m/zratio = 89). The lack of removal of the interferences is due to the fact that in SQ mode there is no barrier present before the reaction cell to hinder 89 Y + and 70 Ge + from entering the cell. In the case of the MS/MS-CH 3 F-As method, accurate results were obtained for both (4 and 5), which is due to the fact, that in the MS/MS mode, the interfering ions 89 Y + and 70 Ge are removed by Q1, (Q1 = 75), which only lets ions through which are on-mass with the m/z-ratio 75. Finally, for the simulated matrix containing 5 μg/l As and 10 μg/l Zn (6) both the SQ-CH 3 F-As method and the MS/MS-CH 3 F-As method lead to accurate results. Thus, the reaction product ion 70 Zn 19 F + is apparently not an interference for neither of the methods under the investigated conditions, which can either be due to the fact that the reaction product ion is not formed in the reaction cell or due to the use of a too low concentration of Zn in the experiment. Thus, only when using MS/MS mode with CH 3 F as reaction gas, it was possible to obtain 5 μg/l As for all simulated matrices. Thus, it was the only method, which could overcome all investigated interferences. Figure 27 is a schematic representation, showing how the different interferences are overcome with the MS/MS-CH 3 F-As method for the interference-free determination of 75 As as 75 AsCH + 2. Figure 27 Schematic representation of the operating principle of the Agilent 8800 instrument when using the MS/MS-CH 3 F- As method, leading to an interference-free determination of 75 As as 75 AsCH 2 +. Modification of figure from [35]. Page 46 of 65

4.5.2 Se Also for Se, simulated matrices were investigated, to demonstrate that the MS/MS-CH 3 F-Se method is able to overcome interferences that other methods, such as He and SQ mode using CH 3 F, fail to overcome. As for As, this was done by investigating how the methods deal with the most important interferences that are present at the original mass of the analyte and thus could interfere with the measurement of 77 Se +, 78 Se + and 80 Se +. However, it was not necessary to investigate the strongest type of interference, 40 Ar 40 Ar + interfering with the determination of 80 Se + and 40 Ar 38 Ar + interfering with the determination of 78 Se +. This interference is always present due to the use of argon as carrier gas and it was already investigated before during method development, where it was possible to check in the blank if this interference was present. No additional interferences were investigated for 80 Se, since it was chosen not to monitor 80 Se in the He method and likewise, no studies of the MS/MS mode using no gas was conducted to protect the detector, as mentioned before. Additionally, potential interferences introduced, when a reaction product is measured at masses 91, 92 and 94 instead of the original ion mass when using CH 3 F as reaction gas, were also investigated. Table 10 lists the interferences investigated using simulated matrices. Table 10 List of on-mass and mass-shift interferences investigated using simulated matrices for Se. Type of interference Mass (amu) Interference 40 Ar 37 Cl +, 40 Ca 37 Cl + 77 On-mass interference 154 Sm 2+, 154 Gd 2+ 78 91 Mass-shift interference due to the use of CH 3 F as reaction gas 92 + forming the product SeCH 2 94 156 Gd 2+ 91 Zr +, 72 Ge 19 F + 92 Zr +, 92 Mo +, 73 Ge 19 F + 94 Zr +, 94 Mo +, 75 As 19 F + In the MS/MS mode, using the gases CH 3 F and He, the following simulated matrices were investigated: 5 μg/l Se standard solution with 500 mg/l Cl, with 500 mg/l Cl and 100 μg/l Ca, with 1 μg/l Sm and with 10 μg/l Gd. Additionally, simulated matrices containing 5 μg/l Se standard solution with 5 μg/l Zr, with 0.1 μg/l Mo, with 0.1 μg/l Ge and with 10 μg/l As were investigated in the MS/MS mode and in the SQ mode using CH 3 F as reaction gas. As for As, the concentrations of the elements were selected such that the interference would be observed, but not more than that. In order to demonstrate that it is possible to obtain accurate results for a pure Se standard (no matrix), a 5 μg/l Se standard solution was also measured in all modes. All samples contained 10 μg/l Te, which was used as internal standard (using the isotope 125 Te). Tellurium was selected as internal standard, since its mass is not too far from the masses of the Se - isotopes and it Page 47 of 65

[Se] (μg/l) has a similar ionization potential, such that is can be used to correct for signal suppression and/or enhancement. Figure 28 shows the results obtained when measuring the simulated matrices in the different MS/MS modes and by using external calibration, whereas figure 29 shows the results obtained when measuring the simulated matrices using CH 3 F as reaction gas, in the MS/MS mode and in the SQ mode and by using external calibration. For each of the simulated matrices, only the isotopes expected to suffer from interference, due to the presence of a particular element in the sample, are shown. The red line indicates the expected concentration of Se at 5μg/L, as present in all solutions. 15 CH3F, 3 F 77 91 10 CH3F, 3 F 78 92 CH3F, 3 F 80 94 (1) (2) (3) (4) (5) He, 77 77 5 He, 78 78 0 5 μg/l Se 5 μg/l Se + 500 mg/l Cl 5 μg/l Se + 500 mg/l Cl + 100 μg/l Ca 5 μg/l Se + 1 μg/l Sm 5 μg/l Se + 10 μg/l Gd Figure 28 Simulated matrices. Se concentration measured with MS/MS-CH 3 F-Se, 77 91 (dark blue), 78 92 (red), 80 94 (green), and with MS/MS-He-As, 77 77 (purple), 78 78 (light blue) is plotted against the different simulated matrices. (n = 5) Figure 28 shows that for all MS/MS methods and for all isotopes, it was possible to obtain accurate results for Se when no extra matrix elements were added (1). When the simulated matrix contained 5 μg/l Se and 500 mg/l Cl (2) and 500 mg/l Cl and 100 μg/l Ca (3), respectively, both methods led to accurate results for Se. As was the case for As, the polyatomic interferences 40 Ar 37 Cl + and 40 Ca 37 Cl + on 77 Se + were removed by energy loss through collision and subsequent kinetic energy discrimination with the MS/MS-He-Se method, (77 77), and with the MS/MS-CH 3 F-Se method, (77 91), the polyatomic interference could be overcome by using the mass-shift method, (Q2 = 91), where 77 Se is measured at mass 91 as 77 SeCH 2 +. In the case where the simulated matrices contained 5 μg/l Se and 1 μg/l Sm (4) and 10 μg/l Gd (5), respectively, it was seen that more than 5 μg/l Se was obtained using the MS/MS-He-Se Page 48 of 65

[Se] (μg/l) method, (77 77) and (78 78). This is because the MS/MS-He-Se method, (77 77) is not able to overcome the interference from the doubly charged interfering 154 Sm 2+ ion. The MS/MS-He- Se method, (77 77 and 78 78), is also not able to overcome the doubly charged interference from 154,156 Gd 2+, where it can be seen that the interference is more obvious for the 78 Se isotope than for the 77 Se isotope, which is caused by the fact that 154 Gd (2.18 %) is less abundant than 156 Gd (20.47 %). The same was seen for the As method using He as collision gas, where this method was also not able to overcome the interference from the doubly charged ions. However with the MS/MS-CH 3 F-Se method, (77 91, 78 92), 5 μg/l Se was again found for both (4 and 5), which again is owing to the successful use of the mass-shift mode. 20 MS/MS, 77 91 15 (2) (3) (4) MS/MS, 78 92 MS/MS, 80 94 10 (1) (5) SQ, 91 SQ, 92 SQ, 94 5 0 5 μg/l Se 5 μg/l Se + 5μg/L Zr 5 μg/l Se + 0.1 μg/l Mo 5 μg/l Se + 0.1 μg/l Ge 5 μg/l Se + 10 μg/l As Figure 29 Simulated matrices. Se concentration measured with MS/MS-CH 3 F-Se, 77 91 (dark blue), 78 92 (red), 80 94 (green), with SQ-CH 3 F-Se, 91 (purple), 92 (light blue), 94 (orange) is plotted against the different simulated matrices. (n = 5) From figure 29, it can be seen than when no additional matrix elements were present (1), it was possible to obtain 5 μg/l Se with all methods relying on CH 3 F as reaction gas in MS/MS and SQ mode. However, in the case of the simulated matrix containing 5 μg/l Se and 5 μg/l Zr (2) and 0.1 μg/l Mo (3), it is seen that with the SQ-CH 3 F-Se method, for none of the isotopes, accurate results were obtained, because of the remaining interference from 91, 92, 92 Zr + (2) and from 92,94 Mo + (3), since these ions overlap with the mass of the reaction product ions formed in the reaction cell, 77 SeCH 2 +, 78 SeCH 2 + and 80 SeCH 2 +, respectively (m/z-ratio = 91, 92 and 94). This method is unable to overcome the interference since, in the SQ mode, all positive ions emerging from the plasma enter the reaction cell. With the MS/MS-CH 3 F-Se method, 5 μg/l Se was obtained, since the interfering ions, 91,92,94 Zr + (2) and 92,94 Mo + (3) are removed by Q1, (Q1 = 77, 78 and 80). Page 49 of 65

Finally, for the simulated matrices containing 5 μg/l Se with 0.1 μg/l Ge (4) and 10 μg/l As (5), respectively, accurate results were not obtained with the SQ-CH 3 F-Se method, which is due to the method not being able to remove the interference from 72,73 Ge 19 F + (4) and from 75 As 19 F + (5), which are formed in the reaction cell due to F atom transfer from the reaction gas CH 3 F, and the reaction products thus formed again overlap with the masses of the Se reaction product ions formed in the reaction cell, 77 SeCH + 2, 78 SeCH + 2 and 80 SeCH + 2, respectively (m/z-ratio = 91, 92 and 94). This is due no barrier being present before the reaction cell to avoid 72,73 Ge + and 75 As + from entering the reaction cell. With the MS/MS-CH 3 F-Se method, 5 μg/l Se was again found for both (4 and 5). This was, as was also the case for As, a result of Q1 being set at the mass of the original analytes (78, 78 and 80, respectively), whereby the ions 72,73 Ge + and 75 As + were removed. Thus, it was successfully demonstrated that the method using CH 3 F as reaction gas in the MS/MS mode was the only method, which allowed for removal of all the interferences investigated. In figure 30, a schematic representation shows how the different interferences are removed with the MS/MS-CH 3 F-Se method for the interference-free determination of 77 Se, 78 Se and 80 Se as 77 SeCH + 2, 78 SeCH + 2 and 80 SeCH + 2, respectively. Page 50 of 65

A) B) C) Figure 30 Schematic representation of the operating principle of the Agilent 8800 instrument when using the MS/MS-CH 3 F- Se method, leading to an interference-free determination of 77 Se (A), 78 Se (B) and 80 Se (C) as 77 SeCH 2 +, 78 SeCH 2 + and 80 SeCH 2 + respectively. Modification of figure from [35]. Page 51 of 65

4.6 Results obtained for reference materials - As and Se As a final validation, the methods for ICP-MS/MS using CH 3 F as reaction gas, MS/MS-CH 3 F-As and MS/MS-CH 3 F-Se, were used to investigate 10 certified reference materials for As and 7 certified reference materials for Se. The certified reference materials investigated are listed in the experimental section, table 4. For the determination of As, the digested samples could be diluted 40- fold, while only a 20-fold dilution was possible for the Se-determination, due to the higher LODs for the Se-method. For both methods, Te was used as internal standard (using the isotope 125 Te) and an external calibration curve was used in order to obtain the final concentrations. Te was chosen as internal standard for both methods, instead of Se, which was used as internal standard when measuring the simulated matrices for As, due to the fact that in several of the reference materials Se is present, and when choosing an internal standard it is important that it is not already present in the matrix. Additionally, Te has an ionization energy of 9.01 ev, thus it is similar to that of As and Se, so it is expected that it is able to correct for the carbon effect (mentioned earlier in section 1) and other matrix effects. In table 11 and table 12, the experimental values, (n = 20), obtained for the certified reference materials for As and Se, respectively, are shown along with the standard deviation (s), relative standard deviation (RSD), the 95 % confidence interval (CI 95 %) and the certified values. The experimental values were obtained from measuring two different digested samples, two times on different days and each measurement consisted of five consecutive measurements for all reference materials (except for urine, which was not a digested sample). For urine, four times five consecutive measurements over two days were performed on samples prepared from the same reconstituted solution. In appendix 4 and 5, the results for the different measurements, (n = 5), can be found. Page 52 of 65

Table 11 Results obtained for the certified reference materials: the experimental values, standard deviation (s), relative standard deviation (RSD), the 95 % confidence interval (CI 95 %) and the certified values for As. (n = 20) Analyte: As Experimental value (μg/g) s (μg/g) RSD (%) CI 95 % (μg/g) Certified value (μg/g) NBS SRM 1575 Pine needles 0.2434 0.0063 2.6 0.0029 0.21 ± 0.04 NBS SRM 1573 Tomato leaves 0.3145 0.0130 4.1 0.0061 0.27 ± 0.05 NIST SRM 1568a Rice flour 0.2835 0.0097 3.4 0.0045 0.29 ± 0.03 CRM 526 Tuna fish tissue 4.954 0.068 1.4 0.032 4.8 ± 0.3 NRC-CNRC DORM-4 Fish protein 6.686 0.059 0.9 0.028 6.80 ± 0.64 BRC 414 Plankton 6.895 0.127 1.8 0.059 6.82 ± 0.28 NBS SRM 1646 Estuarine sediment 10.59 0.28 2.6 0.13 11.6 ± 1.3 NIST SRM 1566a Oyster tissue 13.79 0.19 1.4 0.09 14.0 ± 1.2 NRC-CNRC TORT-3 Lobster Hepatopancreas 66.94 0.41 0.61 0.19 59.5 ± 3.8 Seronorm TM Trace elements Urine, Level 1, Sero, Norway a Unit for reference material, (μg/l), since it is not a solid. 84.71 a 0.82 a 0.97 0.38 a 79 ± 16 a It can be seen from table 11 that when comparing the experimental values to the certified values for As, no significant differences at a 95 % confidence level were found between the experimental values and certified values for the certified reference materials, except for the certified reference material NRC-CNRC TORT-3. Thus the accuracy of the method is good. As previously mentioned, a significant difference is observed, at a 95 % confidence level, for the certified reference material NRC-CNRC TORT-3, since the confidence intervals do not overlap. Different digestions were prepared to try to resolve this, but similar results were obtained for different digestions. Additionally, the reference material was also measured using the Element XR SF-ICP-MS, (n=10), and the experimental value obtained was 67.8 ± 1.9 μg/g, which is in good agreement with the result obtained using the MS/MS-CH 3 F-As method at a 95 % confidence level. Thus, the problem either lies in the digestion process or there is a problem with the reference material caused by contamination. This was however not further investigated due to lack of time. Furthermore, it can be seen that the experimental values obtained for the certified reference materials are both below and above the certified values, thus there is no tendency to always obtain values that are higher or lower than the certified value with the method, which is good. Additionally, from the table it can be seen that the standard deviations on the experimental values are low, which can better be seen by looking at the RSD, which range between 0.61-4.1 %, so even for low values of As, the RSD is low. This means that the precision of the method, a measure for how close the measurements are to one another [47], is good. Additionally it also shows that the reproducibility of the method, thus how close results of repeated measurements of a couple of Page 53 of 65

samples, prepared from the same material and by the same procedure, are to each other [47], is good, since the results are obtained by measuring 2 digestions of the same material on two different days. Table 12 Results obtained for the certified reference materials and the certified values for Se. (n = 20) Analyte: Se NIST SRM 1568a Rice flour NBS SRM 1646 Estuarine sediment BRC 414 Plankton NIST SRM 1566a Oyster tissue NRC-CNRC DORM-4 Fish protein NRC-CNRC TORT-3 Lobster Hepatopancreas Seronorm TM Trace elements Urine, Level 1, Sero, Norway a Non-certified concentration of constituent element b Unit for reference material, (μg/l), since it is not a solid Experimental RSD CI 95 % Isotope s (μg/g) value (μg/g) (%) (μg/g) 77 Se 0.352 0.039 10.9 0.018 78 Se 0.3528 0.0199 5.6 0.0093 80 Se 0.3513 0.0104 3.0 0.0049 77 Se 0.638 0.041 6.4 0.019 78 Se 0.634 0.032 5.0 0.015 80 Se 0.6543 0.0196 3.0 0.0092 77 Se 1.753 0.085 4.9 0.040 78 Se 1.721 0.046 2.7 0.022 80 Se 1.771 0.027 1.5 0.013 77 Se 2.233 0.091 4.1 0.043 78 Se 2.215 0.055 2.5 0.026 80 Se 2.224 0.036 1.6 0.017 77 Se 3.647 0.097 2.7 0.046 78 Se 3.643 0.066 1.8 0.031 80 Se 3.626 0.032 0.87 0.015 77 Se 11.03 0.22 2.0 0.10 78 Se 11.087 0.091 0.82 0.043 80 Se 11.073 0.115 1.0 0.054 77 Se 16.98 b 0.94 b 5.6 0.44 b 78 Se 16.36 b 0.61 b 3.7 0.29 b 80 Se 16.87 b 0.28 b 1.7 0.13 b Certified value (μg/g) 0.38 ± 0.04 (0.6) a 1.75 ± 0.10 2.21 ± 0.24 3.56 ± 0.34 10.9 ± 1.0 13.9 ± 2.8 b Also for Se, it can be seen from table 12, that, at a 95 % confidence level, no significant differences were found between the experimental values and certified values for all certified reference materials. Thus, the accuracy of this method is also good. Furthermore, it can be seen that for all reference materials for Se, the standard deviations and the RSDs, which range between 0.82-10.9 %, are acceptable. This again means that the precision and the reproducibility of this method were good. It can be seen that s and RSD are in general higher for 77 Se followed by 78 Se, while the results based on 80 Se show the lowest s values and RSDs. This is due to the higher sensitivity for 80 Se, followed by that for 78 Se and 77 Se. Page 54 of 65

Furthermore, from the experimental values it can be seen, that both lower and higher values are found in comparison to the certified values, which again is good, since if this tendency was present, this could indicate that a systematic error was present. Additionally, it can be seen that the same results are obtained for the 3 isotopes, thus this opens up possibilities for performing isotopic analysis. Due to lack of time, this was however not further investigated, but it would be interesting to investigate in a future project. Page 55 of 65

5. Conclusion In this project, methods for an interference-free determination of As and Se at ultra-trace levels using the ICP-MS/MS technique with CH 3 F as reaction gas have been successfully developed, optimized and validated. This was feasible due to the possibility of performing a product ion scan with the ICP-MS/MS technique, which made it possible to easily identify reaction products, something which is more difficult with the traditional Q-ICP-MS systems. It was shown that a low LOD could be obtained for both target elements, obtaining the lowest LOD that has ever been obtained for As to the knowledge of the author and obtaining LODs for Se similar to those reported for SF-ICP-MS and it is believed that the LODs for Se could be improved further if higher flows of CH 3 F could be used. An evaluation of the methods using simulated matrices demonstrated that it was possible to overcome interferences that typically cannot be removed with other quadrupole-based methods and to overcome new interferences, which could interfere at the mass of the reaction product ion, due to use of the mass-shift mode. Furthermore, it was possible to determine Se using the three isotopes 77,78,80 Se, where e.g., the isotope 80 Se, the most abundant isotope, is typically not accessible via SF-ICP-MS, since this technique is not able to completely resolve the argon-based interference. This result is also promising in the context of isotopic analysis. Thus, the methods developed match, and in some cases even exceed the capabilities of SF-ICP-MS, which typically is the method of choice for the determination of As and Se. As a proof of concept, the methods were successfully used for the determination of As and Se in reference materials of various biological and environmental origin and similar results were obtained for the three Se - isotopes investigated. Outlook In future work, it would be interesting to apply the methods to real samples and to develop methods where speciation of As and Se species is possible, since, as mentioned in the introduction, different species of As and Se have different toxicity. Additionally it would be interesting to develop a method for simultaneous determination of As and Se. Page 56 of 65

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7. Appendix Appendix 1: All parameters and instrument settings used for the methods As: MS/MS-CH 3 F-As, MS/MS-no gas-as, MS/MS-He-As and SQ-CH 3 F-As and Se: MS/MS-CH 3 F-Se, MS/MS-He-Se and SQ-CH 3 F-Se. Analyte: As Analyte: Se CH 3 F No gas He CH 3 F/He He Scan type MS/MS or SQ MS/MS MS/MS MS/MS or SQ MS/MS Plasma mode Low matrix Low matrix Low matrix Low matrix Low matrix RF power 1550 W 1550 W 1550 W 1550 W 1550 W RF Matching 1.80 V 1.80 V 1.80 V 1.80 V 1.80 V Sample Depth 5.5 mm 5.5 mm 8.0 mm 4.5 mm 4.5 mm Carrier gas 1.18 L/min 1.18 L/min 1.05 L/min 1.13 L/min 1.13 L/min Nebulizer pump 0.20 rps 0.20 rps 0.10 rps 0.20 rps 0.20 rps S/C Temp 2 C 2 C 2 C 2 C 2 C Extract 1-3.0 V -3.0 V 0.0 V -3.9 V -3.9 V Extract 2-185.0 V -175.0 V -195.0-195.0 V -195.0 V Omega Bias -100 V -95 V -105 V -95 V -105 V Omega Lens 9.4 V 10.2 V 9.6 V 11.3 V 11.5 V Q1 entrance 1 V 0 V -5 V -4 V -12 V Q1 Exit 0 V -7 V -1 V 0 V -1V Cell focus 2.0 V -1.0 V 0.0 V 4.0 V 0.0 V Cell Entrance -48 V -46 V -46 V -48 V -50 V Cell Exit -60 V -60 V -70 V -56 V -70 V Deflect 4.0 V 12.8 V -2.2 V 2.6 V -3.2 V Plate Bias -60 V -45 V -60 V -60 V -60 V Q1 mass gain 126 126 126 126 126 Q1 mass offset 126 126 126 126 126 Q1 axis gain 0.9989 0.9989 0.9989 0.9989 0.9989 Q1 axis offset 0.11 0.11 0.11 0.11 0.11 Q1 Bias -2.0 V -1.0 V 0.0 V -1.0 V 0.0 V Q1 Prefilter Bias -14.0 V -18.0 V -18.0 V -20.0 V -12.0 V Q1 Postfilter -22.0 V -34.0 V -20.0 V -38.0 V -38.0 V Reaction gas flow rate 72 % - 4.0 ml/min He 100 % 4.0 ml/min He Octopole Bias -4.1 V -4.1 V -18.0 V -4.1 V -18.0 V Octopole RF 140 V 150 V 160 V 190 V 180 V Energy discrimination -8.4 V -8.4 V 5.0 V -8.4 V 5.0 V Q2 mass gain 128 128 128 128 128 Q2 mass offset 127 127 127 127 127 Q2 axis gain 1.0001 1.0001 1.0001 1.0001 1.0001 Q2 axis offset -0.01-0.01-0.01-0.01-0.01 Q2 QP Bias -12.5 V -12.5 V -13.0 V -12.5 V -13.0 V Torch H 0.0 mm 0.0 mm 0.0 mm 0.2 mm 0.2 mm Torch V 0.2 mm 0.2 mm 0.2 mm 0.2 mm 0.2 mm Discriminator 4.5 mv 4.5 mv 4.5 mv 4.5 mv 4.5 mv Analog HV 1651 V 1651 V 1651 V 1662 V 1662 V Pulse HV 1059 V 1059 V 1059 V 1072 V 1072 V Q1 Q2 77 91 91 77 77 75 89 89 75 75 75 75 78 92 92 78 78 78 78 78 78 78 78 78 80 94 94 80 80 125 125 125 125 125 125 125 Wait time offset 2 ms 2 ms 2 ms 2 ms 2 ms Nr. Replicates 10 10 10 10 10 Nr. Sweep replicates 100 100 100 100 100 Integration time 1 s 1 s 1 s 1 s 1 s Page 61 of 65

Appendix 2: Calibration curves for As: MS/MS-CH 3 F-As, MS/MS-no gas-as, MS/MS-He-As and SQ- CH 3 F-As and Se: MS/MS-CH 3 F-Se, MS/MS-He-Se and SQ-CH 3 F-Se, as well as for Element XR, SF-ICP- MS. (n = 10) Page 62 of 65

Appendix 3: Calibration curves for MS/MS-CH 3 F-As and MS/MS-CH 3 F-Se using 6 % MeOH and 94% HNO 3. (n = 10) Page 63 of 65

Appendix 4: Results obtained for the certified reference material for As, where 1-4 indicate different analyses, each consisting of 5 consecutive measurements. (n = 5) Analyte: As NBS SRM 1575 Pine needles NBS SRM 1573 Tomato leaves NIST SRM 1568a Rice flour CRM 526 Tuna fish tissue NRC-CNRC DORM-4 Fish protein BRC 414 Plankton NBS SRM 1646 Estuarine sediment NIST SRM 1566a Oyster tissue NRC-CNRC TORT-3 Lobster Hepatopancreas Range (μg/g) Exp. value (μg/g) s (μg/g) RSD (%) CI (μg/g) 1 0.2337-0.2564 0.2434 0.0083 3.4 0.0103 2 0.2443-0.2522 0.2473 0.0030 1.2 0.0037 3 0.2312-0.2438 0.2374 0.0047 2.0 0.0058 4 0.2390-0.2493 0.2452 0.0045 1.8 0.0056 1 0.2994-0.3142 0.3066 0.0053 1.7 0.0066 2 0.2990-0.3113 0.3041 0.0053 1.7 0.0066 3 0.3078-0.3242 0.3167 0.0060 1.9 0.0074 4 0.310-0.342 0.331 0.013 4.0 0.016 1 0.2747-0.2934 0.2833 0.0092 3.2 0.0114 2 0.2797-0.2981 0.2886 0.0074 2.6 0.0092 3 0.2834-0.2986 0.2904 0.0060 2.1 0.0074 4 0.2683-0.2756 0.2718 0.0027 1.0 0.0034 1 4.835-4.940 4.879 0.038 0.78 0.048 2 4.925-5.030 4.965 0.044 0.89 0.055 3 4.862-5.064 4.957 0.072 1.5 0.090 4 4.975-5.072 5.015 0.038 0.76 0.048 1 6.618-6.728 6.675 0.050 0.74 0.062 2 6.577-6.813 6.663 0.091 1.4 0.113 3 6.634-6.776 6.714 0.051 0.76 0.064 4 6.663-6.750 6.694 0.038 0.56 0.047 1 6.973-7.089 7.058 0.049 0.70 0.061 2 6.715-6.900 6.815 0.079 1.2 0.098 3 6.828-6.967 6.907 0.051 0.74 0.063 4 6.67-6.92 6.80 0.11 1.6 0.14 1 10.771-10.923 10.836 0.071 0.66 0.089 2 10.621-10.867 10.747 0.087 0.81 0.108 3 10.01-10.31 10.18 0.12 1.2 0.15 4 10.39-10.85 10.59 0.18 1.7 0.23 1 13.66-13.97 13.79 0.14 1.0 0.17 2 13.543-13.621 13.577 0.035 0.26 0.044 3 13.67-14.16 13.93 0.20 1.4 0.25 4 13.71-14.08 13.87 0.17 1.2 0.21 1 66.58-66.88 66.73 0.11 0.16 0.14 2 66.19-67.12 66.61 0.33 0.50 0.42 3 67.03-67.74 67.38 0.29 0.43 0.36 4 66.43-67.37 67.03 0.37 0.55 0.46 Certified value (μg/g) 0.21 ± 0.04 0.27 ± 0.05 0.29 ± 0.03 4.8 ± 0.3 6.80 ± 0.64 6.82 ± 0.28 11.6 ± 1.3 14.0 ± 1.2 59.5 ± 3.8 Seronorm TM Trace elements Urine, Level 1, Sero, Norway a Unit for reference material, (μg/l), since it is not a solid. 79 ± 16 a 2 83.96-86.19 a 84.96 a 0.83 a 0.98 1.04 a 3 83.49-85.61 a 84.76 a 0.81 a 0.95 1.00 a 1 84.02-86.77 a 84.98 a 1.10 a 1.3 1.37 a 4 83.96-84.41 a 84.13 a 0.20 a 0.24 0.25 a Page 64 of 65

Appendix 5: Results obtained for the certified reference material for Se, where 1-4 indicate different analyses, each consisting of 5 consecutive measurements. (n = 5) Analyte: Se NIST SRM 1568a rice flour NBS SRM 1646 Estuarine sediment BRC 414 Plankton NIST SRM 1566a Oyster tissue NRC-CNRC DORM-4 Fish protein NRC-CNRC TORT-3 Lobster Hepatopancreas SeronormTM Trace elements Urine, Level 1, ref. 9067 - Sero, Norway b Range (μg/g) Exp. Value (μg/g) 77 Se s (μg/g) RSD (%) CI 95 % (μg/g) Range (μg/g) Exp. Value (μg/g) 78 Se s (μg/g) RSD (%) CI 95 % (μg/g) Range (μg/g) 1 0.337-0.397 0.380 0.027 7.0 0.033 0.3290-0.3492 0.3381 0.0079 2.3 0.0098 0.3462-0.3654 0.3593 0.0077 2.1 0.0095 2 0.329-0.415 0.367 0.036 9.8 0.045 0.331-0.361 0.345 0.012 3.5 0.015 0.323-0.350 0.343 0.012 3.4 0.014 3 0.293-0.379 0.339 0.034 10.1 0.042 0.326-0.401 0.362 0.027 7.6 0.034 0.3415-0.3681 0.3544 0.0094 2.7 0.0117 4 0.260-0.349 0.323 0.037 11.5 0.046 0.347-0.381 0.365 0.016 4.5 0.020 0.3418-0.3600 0.3484 0.0073 2.1 0.0091 1 0.644-0.695 0.671 0.021 3.1 0.026 0.599-0.669 0.633 0.029 4.6 0.036 0.634-0.688 0.657 0.023 3.5 0.028 2 0.529-0.656 0.615 0.051 8.4 0.064 0.597-0.660 0.637 0.024 3.8 0.030 0.628-0.680 0.653 0.020 3.1 0.025 3 0.586-0.652 0.616 0.027 4.3 0.033 0.583-0.667 0.615 0.031 5.1 0.039 0.636-0.694 0.662 0.021 3.2 0.027 4 0.605-0.696 0.648 0.038 5.8 0.047 0.600-0.707 0.652 0.039 5.9 0.048 0.621-0.658 0.645 0.015 2.3 0.018 1 1.632-1.815 1.737 0.076 4.4 0.094 1.652-1.827 1.736 0.063 3.6 0.078 1.729-1.801 1.772 0.030 1.7 0.037 2 1.701-1.791 1.730 0.036 2.1 0.045 1.666-1.771 1.723 0.038 2.2 0.047 1.724-1.804 1.759 0.030 1.7 0.037 3 1.58-1.95 1.80 0.14 8.0 0.18 1.665-1.747 1.699 0.031 1.8 0.039 1.751-1.823 1.790 0.027 1.5 0.033 4 1.696-1.827 1.750 0.058 3.3 0.072 1.667-1.791 1.726 0.053 3.1 0.066 1.730-1.778 1.763 0.019 1.1 0.024 1 2.12-2.40 2.23 0.11 5.1 0.14 2.163-2.348 2.243 0.070 3.1 0.087 2.181-2.284 2.246 0.043 1.9 0.054 2 2.15-2.45 2.25 0.12 5.2 0.14 2.212-2.270 2.239 0.025 1.1 0.032 2.208-2.289 2.234 0.032 1.4 0.040 3 2.157-2.310 2.230 0.059 2.7 0.074 2.149-2.286 2.203 0.061 2.8 0.075 2.184-2.255 2.224 0.028 1.2 0.034 4 2.075-2.331 2.220 0.095 4.3 0.117 2.135-2.213 2.173 0.034 1.6 0.043 2.178-2.213 2.191 0.015 0.67 0.018 1 3.538-3.798 3.645 0.097 2.7 0.120 3.592-3.792 3.657 0.079 2.2 0.098 3.594-3.669 3.638 0.027 0.76 0.034 2 3.604-3.725 3.687 0.051 1.4 0.064 3.635-3.697 3.670 0.023 0.63 0.029 3.574-3.671 3.614 0.037 1.0 0.046 3 3.53-3.79 3.64 0.11 3.1 0.14 3.595-3.747 3.673 0.064 1.8 0.080 3.590-3.682 3.631 0.035 0.98 0.044 4 3.41-3.75 3.62 0.13 3.7 0.16 3.504-3.614 3.574 0.043 1.2 0.053 3.575-3.652 3.623 0.031 0.86 0.039 1 10.74-11.38 11.20 0.26 2.3 0.32 11.004-11.178 11.112 0.065 0.59 0.081 11.157-11.257 11.211 0.036 0.32 0.045 2 11.00-11.38 11.14 0.16 1.4 0.20 10.86-11.24 11.08 0.14 1.3 0.17 10.982-11.124 11.033 0.057 0.52 0.071 3 10.82-11.04 10.90 0.10 0.93 0.13 10.941-11.161 11.057 0.081 0.73 0.100 10.992-11.173 11.057 0.071 0.64 0.088 4 10.63-11.02 10.88 0.15 1.4 0.19 11.024-11.230 11.099 0.084 0.75 0.104 10.82-11.20 10.99 0.14 1.2 0.17 1 15.17-17.112 16.26 0.76 4.7 0.94 15.66-16.77 16.47 0.46 2.8 0.57 16.52-17.21 16.80 0.26 1.5 0.32 2 15.52-18.38 17.11 1.04 6.1 1.30 16.00-17.77 16.65 0.69 4.1 0.85 16.39-17.55 17.03 0.46 2.7 0.57 3 15.92-17.49 16.80 0.61 3.6 0.76 15.51-17.312 16.31 0.76 4.7 0.95 16.72-17.02 16.86 0.13 0.74 0.16 4 16.56-18.75 17.74 0.87 4.9 1.08 15.25-16.51 16.02 0.48 3.0 0.59 16.46-17.00 16.81 0.22 1.3 0.28 a Non-certified concentration of constituent element b Unit for reference material, (μg/l), since it is not a solid Exp. Value (μg/g) 80 Se s (μg/g) RSD (%) CI 95 % (μg/g) Certified value (μg/g) 0.38 ± 0.04 (0.6) a 1.75 ± 0.10 2.21 ± 0.24 3.56 ± 0.34 10.9 ± 1.0 13.9 ± 2.8 b Page 65 of 65

Interference-free determination of ultra-trace levels of Arsenic and Selenium using methyl fluoride as reaction gas in ICP-MS/MS E. S. Nissen, E. Bolea-Fernandez, L. Balcaen, and F. Vanhaecke Department of Analytical Chemistry, Ghent University, Belgium Determination of As and Se at ultra-trace levels using ICP-mass spectrometry (ICP-MS) is a challenging task due to the presence of spectral overlap. In this work, the use of methyl fluoride (a mixture of 10 % CH 3 F and 90 % of He) was tested as reaction gas in ICP- MS/MS for its capabilities for accurate and precise determination of As and Se. Using product ion scans, the main reaction product ions were identified (AsCH + 2 and SeCH + 2 ) and relied on to measure As and Se at a different and interference-free m/z-ratio with a limit of detection of 0.5 and 7 ng/l, respectively. To check the potential of the CH 3 F methods for the analysis of samples with a heavy matrix, matrix-matched standard solutions were analyzed, whereby it was proven that the CH 3 F methods allow interferencefree determination of both analytes in all cases. Finally, a set of diverse certified reference materials were measured in order to validate the true potential of the methods. All results were in good agreement with the certified values and/or the values obtained by means of sector-field ICP-MS. Keywords: Arsenic, Selenium, ICP-MS/MS, Methyl fluoride 1. Introduction Arsenic and selenium are present in the environment through both natural and anthropogenic routes as a result of agricultural, industrial and mining activities.[1,2] Both are present in inorganic and organic forms and in various oxidation states (-III, 0, +III, +V for As and +VI, +IV, 0, -II for Se). Arsenic is known to be a toxin, whereas Se is an essential element, but becomes toxic at higher concentrations, while the difference between an appropriate and an excessive concentration is small.[3-5] The different species of As and Se found in the environment have different toxicities and inorganic species are known to be more toxic than organic species.[3,6] Thus, it is important to be able to determine As and Se at ultra-trace levels with high accuracy. Arsenic and selenium have been investigated earlier using a variety of analytical techniques, such as atomic absorption spectrometry (AAS) [7-9], atomic fluorescence spectrometry (AFS) [10-12], and inductively coupled plasma-atomic emission spectrometry (ICP-AES). [13-15] However, inductively coupled plasma-mass spectrometry (ICP-MS) has to be considered as the technique of choice for the determination of As and Se, owing to very low detection limits, a wide linear dynamic range, as well as multi-element and isotopic capabilities.[3,6,16-19] However, the determination of As and Se in a complex matrix using this technique remains a challenge due to the following reasons i) As and Se have high ionization energies (9.82 ev and 9.75 ev, respectively), which means that they are poorly ionized under normal ICP-MS 1

conditions, leading to poor sensitivity for the elements, and ii) As and Se suffer from spectral overlap, as a result of the occurrence of, e.g., isobaric, polyatomic and doubly charged ions. See Table I for a list of possible interferences. TABLE I. As and Se - isotopes with their natural isotopic abundance [20] and the most important isobaric, polyatomic and doubly charged interferences [21,22] (non-restrictive list). Abundance Isobaric Doubly charged Analyte Polyatomic interference (%) interference interference 75 As + 100-40 Ar 35 Cl +, 59 Co 16 O +, 36 Ar 38 ArH +, 38 Ar 37 Cl +, 36 Ar 39 K +, 43 Ca 16 O 2 +, 40 Ar 23 Na 12 C +, 12 C 31 P 16 O 2 +, 40 Ca 35 Cl + 150 Nd 2+, 150 Eu 2+, 150 Sm 2+ 74 Se + 0.89 76 Se + 9.37 77 Se + 7.63 78 Se + 23.77 74 Ge + 37 Cl 37 Cl +, 36 Ar 38 Ar +, 38 Ar 36 S +, 40 Ar 34 S +, 39 K 35 Cl +, 58 Ni 16 O + 148 Sm 2+, 148 Nd 2+ 76 Ge + 40 Ar 36 Ar +, 38 Ar 38 Ar +, 60 Ni 16 O +, 39 K 37 Cl +, 41 K 35 Cl + 152 Sm 2+, 152 Gd 2+ - 38 Ar 39 K +, 61 Ni 16 O +, 59 Co 18 O +, 40 Ar 37 Cl +, 40 Ca 37- Cl + 36 Ar 40 ArH +, 38 Ar 2 H +, 12 C 19 F 14 N 16 O 2 + 154 Sm 2+, 154 Gd 2+ 78 Kr + 38 Ar 40 Ca +, 62 Ni 16 O +, 41 K 37 Cl +, 40 Ar 38 Ar + 156 Gd 2+, 156 Dy 2+ 80 Se + 49.61 80 Kr + 40 Ar 40 Ca +, 64 Ni 16 O +, 64 Zn 16 O +, 32 S 2 16 O +, 32 S 16 O 3 +, 40 Ar 40 Ar +, 40 Ca 40 Ca +, 160 Gd 2+, 160 Dy 2+ 82 Se + 8.73 82 Kr + 40 Ar 42 Ca +, 34 S 16 O 3 +, 66 Zn 16 O +, 12 C 35 Cl 2 +, 40 Ar 2 H 2 + 164 Dy 2+, 164 Er 2+ In literature, a variety of approaches have been proposed to tackle these problems. With regard to the problem of poor ionization, several studies have used the addition of carbon as a mean of increasing the signal intensity, which is mainly due to the charge transfer reaction, where C + species transfer their charge to As and Se atoms, but also due to e.g., an enhancement of the nebulization efficiency for the sample.[23] In literature, enhancements of more than 150 % for As and Se using methanol have been reported.[18] However, the carbon effect can also be a problem in the analysis if the sample contains large amounts of carbon and proper correction, by using a suitable internal standard, by adding organic solvents to both standard and samples or by using standard addition for calibration, is necessary.[24] The problem of spectral overlap has mainly been dealt with in two ways. One option is to use an SF-ICP-MS instrument, where higher mass resolution is used to overcome spectral overlap. However, this instrument comes at a high purchase price and obtaining the highest mass resolution (necessary to determine As and Se), is accomplished with a loss in sensitivity of 2 orders of magnitude.[ 19] Another option is to use a quadrupolebased ICP-MS instrument equipped with a collision/reaction cell, where chemical resolution is used to deal with spectral overlap. Interferences can be removed by collision using, e.g., a gas mixture such as He/H 2, though this also reduces the transmission of As + and Se + [16,19], or by using a reaction gas such as O 2, which can either react with the interfering ion or with the analyte ion, whereby the analyte ion can be determined interference-free in the on-mass mode or in the mass-shift mode, respectively [25,26]. In 2013, a new generation of quadrupole-based ICP-MS instrumentation was introduced, ICP-MS/MS, where an octopole-based collision/reaction cell is placed inbetween two quadrupole mass analyzers. The introduction of the first quadrupole allows only the analyte ions and other on-mass ions to pass to the cell. Thus, the processes taking place in the collision/reaction cell are more under control and additionally, this setup enables the possibility to perform a product ion scan, which can aid in the 2

identification of reaction products formed in the cell. Thus the operation in the MS/MS mode should deal better with spectral overlap and opens up the possibility to use more unconventional reaction gases, such as CH 3 F.[19,27] CH 3 F can react with an ion according to 5 main reaction paths [28] and thus, it is difficult to predict which will be the main reaction path for the different analytes. The main goal of this project is to investigate the capabilities of CH 3 F as reaction gas in ICP-MS/MS to resolve the spectral overlap that As and Se suffer from, with the aim of developing sensitive and selective methods for the determination of As and Se at ultratrace levels in diverse samples. 2.1 Instrumentation 2. Experimental To carry out all measurements, an Agilent 8800 triple quadrupole ICP-MS instrument (ICP-QQQ/Agilent technologies, Japan) was used. The instrument is equipped with an introduction system, comprising of a concentric nebulizer and a Scott-type double-pass spray chamber, and a mass separation device, comprising of two quadrupole mass analyzers with an octopole-based collision/reaction cell fitted in-between (Figure 1). The instrument can be operated in the "vented mode" (no gas in the cell) or the cell can be pressurized with a collision gas (e.g., He) or a reaction gas (e.g., H 2, O 2, NH 3 ) or a mixture of both. Figure 1. Schematic overview of the operation principle of the ICP-MS/MS, where a general example is given for the interference-free determination of X M + as X MCH 2 +, where M = analyte, N = interference, X = mass, Y = mass X. Modification of figure from [27]. In this project a rather unconventional reaction gas was used, CH 3 F/He in a 10/90 % mixture, which was introduced via the 4 rd gas inlet. The gas flow rate is controlled by a mass flow controller, which is calibrated for O 2, and allows a flow of 0-100 %, which is equivalent to 0-1 ml/min O 2. The analytes 75 As and 77, 78, 80 Se were monitored. SF-ICP-MS, which was - until now - the method of choice for the determination of low levels of As and Se, was used for validation purposes. The instrument used is a Thermo Element XR SF-ICP-MS (Thermo-Scientific, Germany). The most important settings and parameters can be found in Table II for the Agilent 8800 ICP-QQQ and in Table III for the Thermo Element XR SF-ICP-MS. 3