INTRODUCTION:

High performance liquid chromatography (HPLC) when coupled to ICP-MS for elemental detection is by far the most widely used technique for speciation analysis. For speciation analysis of environmental samples that are not already in liquid form, three steps are generally required.¹

The first step comprises the extraction of the element species from the sample, because so far, no techniques are available to do in situ speciation analysis at environmental concentrations. During this critical extraction step, the compounds should be quantitatively extracted and analyzed. ICP MS is superior to molecule-selective detection using electrospray ionization. Compound independent quantification not be changed. Combinations of water and/or organic solvents or weak acids are often employed for this purpose. In a second step, the compounds are then separated by liquid chromatography.

After the separation of the compounds, a reliable detection step is necessary. ICP-MS is typically the detector of choice because of its excellent detection limits, wide dynamic range, multi-element capabilities and the possibility to determine isotopes².

The robustness of the ICP is certainly one of the reasons that make the hard ionization in elements should be mentioned as another benefit of ICP-MS. This allows the quantification of compounds without having a standard available.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY:

HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller adsorbent particles (2–50 μm in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.

HPLC TECHNIQUES USED IN HPLC -ICP-MS

Reverse phase HPLC:

One of the major interest for using RP–HPLC for the separation of species of interest prior to ICP–MS detection is the simplicity of the technique. Many applications were found for inductively coupled plasma–mass spectrometry (ICP-MS) detection with reversed-phase high performance liquid chromatography (HPLC) for the speciation of pharmaceutical compounds containing a transition metal (cobalt) or the nonmetallic heteroatoms sulfur, phosphorus, chlorine, and bromine. Multiple elements were monitored simultaneously by ICP-MS in order to fully utilize specificity of elemental detection.

Ion Chromatography:

Metal-free ion chromatography (IC) separates the individual ionic species without contributing trace metal contamination.⁴Ion Chromatography-Inductively Coupled Plasma Mass Spectrometry (IC-ICP-MS) performs trace elemental detection and quantification. Metal-free IC (with high resolution ion exchange columns and simple online connectivity) together with high sensitivity ICP-MS and integrated software are a powerful combination for fast and efficient metal speciation.

Chiral Chromatography (CC):

Chiral separations using chiral stationary phases are based on the formation of transient diastereomeric complexes between the enantiomers and the chiral ligand of the stationary phases. coupling of a crown ether column to ICP–MS for the separation of L, D Se-methionine in Se nutritional supplements has been carried out. Tilcoplanin based column to separate several selenoamino acid enantiometers, and both UV and ICP-MS detections were used and compared in the same injection.

INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS):

Instrumentation:

It is a type of mass spectrometry which is capable of detecting metals and several non-metals at concentrations as low as one part in 10¹⁵ (part per quadrillion, ppq) on non-interfered low-background isotopes. This is achieved by ionizing the sample with inductively coupled plasma and then using a mass spectrometer to separate and quantify those ionsCompared to atomic absorption spectroscopy, ICP-MS has greater speed, precision, and sensitivity.

An inductively coupled plasma is a plasma that is energized (ionized) by inductively heating the gas with an electromagnetic coil, and contains a sufficient concentration of ions and electrons to make the gas electrically conductive. Even a partially ionized gas in which as little as 1% of the particles are ionized can have the characteristics of a plasma (i.e., response to magnetic fields and high electrical conductivity). The plasmas used in spectrochemical analysis are essentially electrically neutral, with each positive charge on an ion balanced by a free electron. In these plasmas the positive ions are almost all singly charged and there are few negative ions, so there are nearly equal amounts of ions and electrons in each unit volume of plasma.

To maximise plasma temperature (and hence ionisation efficiency) and stability, the sample should be introduced through the central tube with as little liquid (solvent load) as possible, and with consistent droplet sizes.

Coupling:

For coupling to mass spectrometry, the ions from the plasma are extracted through a series of cones into a mass spectrometer, usually a quadrupole. The ions are separated on the basis of their mass-to-charge ratio and a detector receives an ion signal proportional to the concentration.

The constituents of ICP-MS instrument are designed to allow for reproducible and/or stable operation.

Plasma torch:

The plasma used in an ICP-MS is made by partially ionizing argon gas (Ar → Ar⁺ + e⁻). The energy required for this reaction is obtained by pulsing an alternating electric current in wires that surround the argon gas⁶.

Transfer of ions into vacuum:

The carrier gas is sent through the central channel and into the very hot plasma. The sample is then exposed to radio frequency which converts the gas into a plasma. The high temperature of the plasma is sufficient to cause a very large portion of the sample to form ions.⁶ This fraction of ionization can approach 100% for some elements (e.g. sodium), but this is dependent on the ionization potential. A fraction of the formed ions passes through a ~1 mm hole (sampler cone) and then a ~0.4 mm hole (skimmer cone). The purpose of which is to allow a vacuum that is required by the mass spectrometer. The vacuum is created and maintained by a series of pumps.

Ion optics:

Before mass separation, a beam of positive ions has to be extracted from the plasma and focused into the mass-analyzer. It is important to separate the ions from UV photons, energetic neutrals and from any solid particles that may have been carried into the instrument from the ICP. Traditionally, ICP-MS instruments have used transmitting ion lens arrangements for this purpose. Examples include the Einzel lens, the Barrel lens, Agilent's Omega Lens⁷and Perkin-Elmer's Shadow Stop⁸. Another approach is to use ion guides (quadrupoles, hexapoles, or octopoles) to guide the ions into mass analyzer along a path away from the trajectory of photons or neutral particles.

If the mass of interest has a low sensitivity and is just below a much larger peak, the low mass tail from this larger peak can intrude onto the mass of interest. A Retardation Filter might be used to reduce this tail. This sits near the collector, and applies a voltage equal but opposite to the accelerating voltage; any ions that have lost energy while flying around the instrument will be decelerated to rest by the filter.

Collision reaction cell and iCRC:

The collision/reaction cell is used to remove interfering ions through ion/neutral reactions⁵. Collision/reaction cells are known under several names. The dynamic reaction cell is located before the quadrupole in the ICP-MS device⁹ The chamber has a quadrupole and can be filled with reaction (or collision) gases (ammonia, methane, oxygen or hydrogen), with one gas type at a time or a mixture of two of them, which reacts with the introduced sample, eliminating some of the interference.

The integrated Collisional Reaction Cell (iCRC) used in ICP-MS is a mini-collision cell installed in front of the parabolic ion mirror optics that removes interfering ions by injecting a collisional gas (He), or a reactive gas (H₂), or a mixture of the two, directly into the plasma as it flows through the skimmer cone and/or the sampler cone. The iCRC removed interfering ions using a collisional kinetic energy discrimination (KED) phenomenon and chemical reactions with interfering ions similarly to traditionally used larger collision cells.

Figure 2: Detailed illustration of ICP-MS system

MASS ANALYZERS USED IN ICP -MS:

Multipole ion guide:

Multipoles ion guides are generally created using metal rods of a certain cross-sectional shape arranged symmetrically around a central axis. The exact number and shape of the rods varies, depending on the desired use. The number of rods is virtually always even, with the most common numbers being 4, 6 and 8. The rod shapes are typically rectangular, round or hyperbolic.⁴ Here are a few examples:

The number of rods is commonly referred to as the number of "poles" of the ion guide. As such, the common names for various numbers of rods/poles, are:

· Four rods: Quadrupole

· Six rods: Hexapole

· Eight rods: Octopole

· Ten rods: Decapole

· Twelve rods: Dodecapole

If we assume that an ion guide's rods are much longer than the field radius and are machined/aligned with high precision, then the electric field within the bulk of the ion guide's length will be uniform. In other words, no matter where the ions are located within the length of the guide, they will always feel the same electric field in the radial (xy) plane. In order to generate a multipole potential, the set of rods is separated into two pairs, alternating as you go around the central axis such that each rod is neighbored by rods of the other pair. Once the rods are grouped into pairs, the first pair has a particular voltage applied to it, while the second pair has an equal voltage of the opposite polarity applied to it⁴.

Figure 3: multipole fields (The guide is broken into two sets of rods with opposing voltages applied. As aDC field would not focus ions)

Double-focusing mass spectrometer:

A mass spectrometer that applies an electric field in order to select ions that have the same kinetic energy from an ion beam and uses a wedge-shaped magnetic field to separate the total ion beam into discrete ion beams according to the mass-to-charge ratio of the ions.

DETECTORS IN ICP-MS:

Electron multiplier tube:

An electron multiplier is a vacuum-tube structure that multiplies incident charges. In a process called secondary emission, a single electron can, when bombarded on secondary-emissive material, induce emission of roughly 1 to 3 electrons. If an electric potential is applied between this metal plate and yet another, the emitted electrons will accelerate to the next metal plate and induce secondary emission of still more electrons. Some advantages of using this include a response time in the picoseconds, a high sensitivity, and an electron again of about 10⁸ electrons¹⁰. This can be repeated a number of times, resulting in a large shower of electrons all collected by a metal anode.

Figure 4 : The continuous electron multiplier tube

Faraday cup detector:

A Faraday cup is a metal (conductive) cup designed to catch charged particles in vacuum The resulting current can be measured and used to determine the number of ions or electrons hitting the cup. Faraday cup can act as a collector for electrons in a vacuum (for instance from an electron beam). In this case electrons simply hit the metal plate/cup and a current is produced¹. Faraday cups are not as sensitive as electron multiplier detectors, but are highly regarded for accuracy because of the direct relation between the measured current and number of ions.This device is considered a universal charge detector because of its independence from the energy, mass, chemistry, etc. of the analyte.

Figure 5: Schematic of faraday cup

APPLICATIONS OF HPLC ICP-MS:

HPLC–ICP–MS has been employed in a wide range of disciplines including environmental, biological, and clinical applications. It represents a broad, multidisciplinary ﬁeld of study, and a number of recent reviews and books provide an excellent reference to the role of various elements in these ﬁelds. Since most studies are related to concerns about human health risks, the majority of the publications have been focusing on only a few toxic element species. About 60% of all papers are dealing with only 5 elements, namely arsenic, selenium, mercury, chromium, and tin. Another 30% of papers are dealing with copper, zinc, lead, cadmium, and iron. All other elements, in total, are in the focus of only 10% of the publications. Some of the more recent, typical, and important applications in these ﬁelds will be cited and discussed in the following sections.

Arsenic Speciation in Rice:

Rice constitutes a major food source in a large part of the world. Unfortunately, rice accumulates arsenic and concentrations up to 2 µg/g dry weight of rice have been reported. The arsenic in rice may be present as either the more toxic inorganic forms, As (III) and As (V), or in the lesser toxic organic forms, mainly dimethylarsinic acid — DMA(V)¹³. A large variation is found in the arsenic speciation of rice depending on one or a combination of factors including the genotype of rice and environmental factors such as soil composition. Identifying the safest variety of rice to consume could potentially minimize the exposure of millions of people to inorganic arsenic. The amount of inorganic arsenic can be calculated as the sum of the As(III) and As(V) peaks in chromatogram.

Antimony Speciation in Natural Waters by HPLC-ICP-MS:

Arsenic and antimony belong to the same family of the periodic table. However, during the last 10 years, a lot of work has been done on arsenic speciation but relatively little on antimony speciation, even though antimony is widely used in a range of industrial and consumer products, which can act as sources of environmental and human exposure. Antimony exists in environmental systems in different oxidation states (-3, 0, +3 and +5). In water, only trivalent and pentavalent oxidation states are found, predominantly Sb(V) ¹⁴

The average concentration of antimony in natural surface water is around 1 µg/L but this can increase to 100 µg/L near a contaminated source. Under reducing conditions, Sb (III) is found in contaminated water. B The toxicity of Sb (III) is greater than the toxicity of Sb(V).

HPLC-ICP-MS for Determination of Methyl-Selenium Metabolites of Relevance to Health in Pharmaceutical Supplements:

Knowledge of speciation of selenium in food and food supplements will have implications with respect to the determination of Se requirements and to the investigation of relationships between Se status and health and disease. It will help in the development of safe and effective products and with future regulation of their production and use. Speciation by reversed-phase (RP) HPLC-ICP-MS A portion of the diluted extract can be analyzed by ion-pairing RP HPLC-ICP-MS at a flow rate of 0.5 mL/ min using a water/methanol (98:2, v/v) mixture containing 0.1% (v/v) formic acid as the mobile phase. The Se concentration of γ-glutamyl-SeMC in the water-soluble extracts is given the average ± SD (n = 3) expressed in the dry sample. Total Se determination of the yeast samples was performed by ICP-MS after microwave acid digestion.

Detection of Heteroatom-Tagged Green Fluorescence Protein by HPLC Photodiode Array (PDA) Detector, and ICP-MS:

Tagging of proteins and metabolites with heteroatoms (for example, sulfur (S), phosphorus (P), selenium (Se), and iodine (I)) and heteroisotopes (enriched stable isotopes) and their detection with an ICP-MS offers several advantages in proteomics and metabolomics. However, since the hitherto techniques for protein tagging were always post-translational modifications, the target proteins were limited to phosphorylated and antibody-available proteins¹⁶. Therefore, on-translational tagging with a heteroisotope in the form of [34S]-enriched Met, or a heteroatom, that is, selenomethionine (SeMet) or telluromethionine (TeMet), could provide an excellent tool for quantitative proteomics

Analysis of Phospholipids:

Phospholipids are the main constituents of membranes in all types of prokaryotic and eukaryotic cells. Due to their complexity and heterogeneity in biological samples, qualitative and quantitative analyses of membrane phospholipids in cellular extracts represent major analytical challenges, mainly due to the requirement for suitable and sensitive detection method. ICP-MS is a suitable detector for selective determination of phospholipids, which all contain phosphorus Phospholipids are extractable with organic solvents; therefore, liquid chromatography with an organic mobile phase was used for separation of different lipid species.

Analysis of Glyphosate, Gluphosinate, and AMPA by Ion-Pairing LC-ICP-MS:

Glyphosate and the related compound gluphosinate are among the most widely used of nonselective herbicides. They act by inhibiting the synthesis of specific amino acids. AMPA (aminomethylphosphonic acid) is the major metabolite. While LC separation of these compounds is fairly straightforward and specific, sensitive detection has been problematic due to poor ionization characteristics in LC/ MS. Detection of phosphorus using collision cell ICP-MS to eliminate the common interferences from NO+ and NOH+ when coupled to HPLC can provide a simple, highly sensitive method of analysis for these compounds¹⁸

Analysis of trace elements in body fluids:

This technique has been applied to the speciation of arsenic in urine with a boric acid buffer mobile phase containing n-propanol as an organic modiﬁer and cetyltrimethylammonium bromide (CTAB) as the surfactant. Such a technique is capable of simultaneously separating anionic and cationic species, the resolution of As(III), As(V), MMA, and DMA was accomplished in less than 15 min. Fe, Cu, and Zn have been simultaneously speciated in human serum by HPLC–ICP–MS using on-line isotope dilution. Separation was performed in an anion-exchange column (Mono-Q HR 5/5) with a mobile phase gradient of ammonium acetate at pH 7.4³

Similarly, zinc binding proteins have been analyzed in human serum with SEC–ICP–MS, as well as anion-exchange chromatography. Breast milk, like any other bodily ﬂuid, also contains trace elements. The speciation of these elements is particularly important since breast milk is the sole source of dietary nutrition for infants and can be extremely valuable in the design of infant formulas. Speciation of all elements of nutritional interest can be done with LC–ICP–M.

ACKNOWLEDGEMENT:

The author would like to express her gratitude to Dr Bhavya Sri and Dr M Sumakanth for their guidance and reviews, grateful to RBVRR Women˚s college of pharmacy for help and support.

REFERENCES:

1. Principles of Instrumental Analysis, Fifth Edition by Skoog. Holler, Nieman.

2. Hand book of ICP-MS Applications. 2^nd Edition by Aglilent Technologies.

3. Tiebang Wang, LC-ICP-MS, journal of liquid chromatography and related technologies, process research and development, Merck Research Laboratories, Rahway, New Jersey, USA,30:807-831, 2007.

4. http://www.massspecpro.com/technology/ion-optics/multipole-ion-guide.

5. Yip,y,sham,W2007.''Applications of collision/reaction-cell technology in isotope dilution mass spectrometry TrAc Trends in Analytical Chemistry.26(7):727.

6. Thomas Robert. A Beginners guide to ICP-MS (pdf) 2014.

7. Kenichi Sakata et al., Inductively coupled plasma mass spectrometer and method, US patent 6265717 B1.

8. Scott D. Tanner et al., Device and method preventing ion source gases from entering reaction cell, US patent 6639665 B2.

9. S. Tanner; V. Baranov; D. Bandura (2002). "Reaction cells and collision cells for ICP-MS: a tutorial review". 57 (9): 1361–1452.

10. Tao, S., Chan, H., and Vander Graaf, H. (2016). Secondary Electron Emission Materials for Transmission Dynodes in Novel Photomultipliers: A Review. Materials, 9(12), 1017.

11. Green field, 1994.Inductively coupled plasma Mass spectrometry.

12. Louri Kalinitchenko Ion optical system for a Mass Spectrometer, United states Patent number6614(2003).

13. Williams, P. N., Price, A. H., Raab, A., Hossain, A., Feldmann, J. and Meharg, A. A. (2005). Variation in arsenic speciation and concentration in paddy rice related to dietary exposure. Environ. Sci. Technol., 39(15), 5531–5540

14. Center of Expertise on Analysis of Environmental compound (2010). Determination of speciation in antimony, and chromium: Methods as per liquid chromatography coupled to inductively coupled plasma mass spectrometry in argon plasma source MA -200.

15. Goenaga-Infante, H., Hearn, R. and Catterick, T. (2005). Anal. Bioanal. Chem., 382, 957–967.

16. Fowler, B. A., Geering, P. L. and Merien, E. (ed.) (1991). Metals and Their Compounds in the Environment: Occurrence, Analysis and Biological Relevance. VCH, New York, USA, 743.V.Barano

17. S. Tanner 1999. A dynamic cell reaction of icp-ms

18. Sas, B., Peys, E. and Helsen, M. (1999). J. Chromatogr. A, 864, 179–182.

19. Lopez-Avila, V., Sharpe, O. and Robinson, W. (2006, September). Determination of ceruloplasmin in human serum by SEC-ICP-MS. Analytical and Bioanalytical Chemistry, 386(1), 180–187.

20. Goenaga-Infante, H. et al. (2005). Journal of Analytical Atomic Spectrometry, 20, 864–870.

Received on 31.05.2019 Modified on 15.06.2019

Asian J. Research Chem. 2019; 12(4):225-230.

DOI: 10.5958/0974-4150.2019.00043.9