Plasma-based instrumental techniques that are useful for pharmaceutical analyses fall into two major categories: those based on the inductively coupled plasma, and those where a plasma is generated at or near the surface of the sample. An inductively coupled plasma (ICP) is a high-temperature excitation source that desolvates, vaporizes, and atomizes aerosol samples and ionizes the resulting atoms. The excited analyte ions and atoms can then subsequently be detected by observing their emission lines, a method termed inductively coupled plasma–atomic emission spectroscopy (ICP–AES), also known as inductively coupled plasma–optical emission spectroscopy (ICP–OES); or the excited or ground state ions can be determined by a technique known as inductively coupled plasma–mass spectrometry (ICP–MS). ICP–AES and ICP–MS may be used for either single- or multi-element analysis, and they provide good general-purpose procedures for either sequential or simultaneous analyses over an extended linear range with good sensitivity.

An emerging technique in plasma spectrochemistry is laser-induced breakdown spectroscopy (LIBS). In LIBS, a solid, liquid, or gaseous sample is heated directly by a pulsed laser, or indirectly by a plasma generated by the laser. As a result, the sample is volatilized at the laser beam contact point, and the volatilized constituents are reduced to atoms, molecular fragments, and larger clusters in the plasma that forms at or just above the surface of the sample. Emission from the atoms and ions in the sample is collected, typically using fiber optics or a remote viewing system, and measured using an array detector such as a charge-coupled device (CCD). LIBS can be used for qualitative analysis or against a working standard curve for quantitative analysis. Although LIBS is not currently in wide use by the pharmaceutical industry, it might be suited for at-line or on-line measurements in a production setting as well as in the laboratory. Because of its potential, it should be considered a viable technique for plasma spectrochemistry in the pharmaceutical laboratory. However, because LIBS is still an emerging technique, details will not be further discussed in this general chapter.1

Sample preparation is critical to the success of plasma-based analysis and is the first step in performing any analysis via ICP–AES or ICP–MS. Plasma-based techniques are heavily dependent on sample transport into the plasma, and because ICP–AES and ICP–MS share the same sample introduction system, the means by which samples are prepared may be applicable to either technique. The most conventional means by which samples are introduced into the plasma is via solution nebulization. If solution nebulization is employed, solid samples must be dissolved in order to be presented into the plasma for analysis. Samples may be dissolved in any appropriate solvent. There is a strong preference for the use of aqueous or dilute nitric acid solutions, because there are minimal interferences with these solvents compared to other solvent choices. Hydrogen peroxide, hydrochloric acid, sulfuric acid, perchloric acid, combinations of acids, or various concentrations of acids can all be used to dissolve the sample for analysis. Dilute hydrofluoric acid may also be used, but great care must be taken to ensure the safety of the analyst, as well as to protect the quartz sample introduction equipment when using this acid; specifically, the nebulizer, spray chamber, and inner torch tube should be manufactured from hydrofluoric acid-tolerant materials. Additionally, alternative means of dissolving the sample can be employed. These include, but are not limited to, the use of dilute bases, straight or diluted organic solvents, combinations of acids or bases, and combinations of organic solvents.

When samples are introduced into the plasma via solution nebulization, it is important to consider the potential matrix effects and interferences that might arise from the solvent. The use of an appropriate internal standard and/or matching the standard matrix with samples should be applied for ICP–AES and ICP–MS analyses in cases where accuracy and precision are not adequate. In either event, the selection of an appropriate internal standard should consider the analyte in question, ionization energy, wavelengths or masses, and the nature of the sample matrix.

Where a sample is found not to be soluble in any acceptable solvent, a variety of digestion techniques can be employed. These include hot-plate digestion and microwave-assisted digestions, including open-vessel and closed-vessel approaches. The decision regarding the type of digestion technique to use depends on the nature of the sample being digested, as well as on the analytes of interest.

Open-vessel digestion is generally not recommended for the analysis of volatile metals, e.g., selenium and mercury. The suitability of a digestion technique, whether open-vessel or closed-vessel, should be supported by spike recovery experiments in order to verify that, within an acceptable tolerance, volatile metals have not been lost during sample preparation. Use acids, bases, and hydrogen peroxide of ultra-high purity, especially when ICP–MS is employed. Deionized water must be at least 18 megaohm. Check diluents for interferences before they are used in an analysis. Because it is not always possible to obtain organic solvents that are free of metals, use organic solvents of the highest quality possible with regard to metal contaminants.

It is important to consider the selection of the type, material of construction, pretreatment, and cleaning of analytical labware used in ICP–AES and ICP–MS analyses. The material must be inert and, depending on the specific application, resistant to caustics, acids, and/or organic solvents. For some analyses, diligence must be exercised to prevent the adsorption of analytes onto the surface of a vessel, particularly in ultra-trace analyses. Contamination of the sample solutions from metal and ions present in the container can also lead to inaccurate results.

The use of labware that is not certified to meet Class A tolerances for volumetric flasks is acceptable if the linearity, accuracy, and precision of the method have been experimentally demonstrated to be suitable for the purpose at hand.

There are two ways to introduce the sample into the nebulizer: by a peristaltic pump and by self-aspiration. The peristaltic pump is preferred and serves to ensure that the flow rate of sample and standard solution to the nebulizer is the same irrespective of sample viscosity. In some cases, where a peristaltic pump is not required, self-aspiration can be used.

A wide variety of nebulizer types is available, including pneumatic (concentric and cross-flow), grid, and ultrasonic nebulizers. Micronebulizers, high-efficiency nebulizers, direct-injection high-efficiency nebulizers, and flow-injection nebulizers are also available. The selection of the nebulizer for a given analysis should consider the sample matrix, analyte, and desired sensitivity. Some nebulizers are better suited for use with viscous solutions or those containing a high concentration of dissolved solids, whereas others are better suited for use with organic solutions.

Note that the self-aspiration of a fluid is due to the Bernoulli, or Venturi, effect. Not all types of nebulizers will support self-aspiration. The use of a concentric nebulizer, for example, is required for self-aspiration of a solution.

Once a sample leaves the nebulizer as an aerosol, it enters the spray chamber, which is designed to permit only the smallest droplets of sample solution into the plasma; as a result, typically only 1% to 2% of the sample aerosol reaches the ICP, although some special-purpose nebulizers have been designed that permit virtually all of the sample aerosol to enter the ICP. As with nebulizers, there is more than one type of spray chamber available for use with ICP–AES or ICP–MS. Examples include the Scott double-pass spray chamber, as well as cyclonic spray chambers of various configurations. The spray chamber must be compatible with the sample and solvent and must equilibrate and wash out in as short a time as possible. When a spray chamber is selected, the nature of the sample matrix, the nebulizer, the desired sensitivity, and the analyte should all be considered.

Gas and liquid chromatography systems can be interfaced with ICP–AES and ICP–MS for molecular speciation, ionic speciation, or other modes of separation chemistry, based on elemental emission or mass spectrometry.

Ultimately, the selection of sample introduction hardware should be demonstrated experimentally to provide sufficient specificity, sensitivity, linearity, accuracy, and precision of the analysis at hand.

In addition to solution nebulization, it is possible to analyze solid samples directly via laser ablation (LA). In such instances, the sample enters the torch as a solid aerosol. LA–ICP–AES and LA–ICP–MS are better suited for qualitative analyses of pharmaceutical compounds because of the difficulty in obtaining appropriate standards. Nonetheless, quantitative analyses can be performed if it can be demonstrated through appropriate method validation that the available standards are adequate.2

Single- or multi-element standard solutions, whose concentrations are traceable to primary reference standards, such as those of the National Institute of Standards and Technology (NIST), can be purchased for use in the preparation of working standard solutions. Alternatively, standard solutions of elements can be accurately prepared from standard materials and their concentrations, determined independently, as appropriate. Working standard solutions, especially those used for ultra-trace analyses, may have limited shelf life. As a general rule, working standard solutions should be retained for no more than 24 hours unless stability is demonstrated experimentally. The selection of the standard matrix is of fundamental importance in the preparation of element standard solutions. Spike recovery experiments should be conducted with specific sample matrices in order to determine the accuracy of the method. If sample matrix effects cause excessive inaccuracies, standards, blanks, and sample solutions should be matrix matched, if possible, in order to minimize matrix interferences.

In cases where matrix matching is not possible, an appropriate internal standard or the method of standard additions should be used for ICP–AES or ICP–MS. Internal standards can also be introduced through a T connector into the sample uptake tubing. In any event, the selection of an appropriate internal standard should consider the analytes in question, their ionization and excitation energies, their chemical behavior, their wavelengths or masses, and the nature of the sample matrix. Ultimately, the selection of an internal standard should be demonstrated experimentally to provide sufficient specificity, sensitivity, linearity, accuracy, and precision of the analysis at hand.

The method of standard additions involves adding a known concentration of the analyte element to the sample at no fewer than two concentration levels plus an unspiked sample preparation. The instrument response is plotted against the concentration of the added analyte element, and a linear regression line is drawn through the data points. The absolute value of the x-intercept multiplied by any dilution factor is the concentration of the analyte in the sample.

The presence of dissolved carbon at concentrations of a small percentage in aqueous solutions enhances ionization of selenium and arsenic in an inductively coupled argon plasma. This phenomenon frequently results in a positive bias for ICP–AES and ICP–MS selenium and arsenic quantification measurements, which can be remedied by using the method of standard additions or by adding a small percentage of carbon, such as analytically pure glacial acetic acid, to the linearity standards.

The components that make up the ICP excitation source include the argon gas supply, torch, radio frequency (RF) induction coil, impedance-matching unit, and RF generator. Argon gas is almost universally used in an ICP. The plasma torch consists of three concentric tubes designated as the inner, the intermediate, and the outer tube. The intermediate and outer tubes are almost universally made of quartz. The inner tube can be made of quartz or alumina if the analysis is conducted with solutions containing hydrofluoric acid. The nebulizer gas flow carries the aerosol of the sample solution into and through the inner tube of the torch and into the plasma. The intermediate tube carries the intermediate (sometimes referred to as the auxiliary) gas. The intermediate gas flow helps to lift the plasma off the inner and intermediate tubes to prevent their melting and the deposition of carbon and salts on the inner tube. The outer tube carries the outer (sometimes referred to as the plasma or coolant) gas, which is used to form and sustain the toroidal plasma. The tangential flow of the coolant gas through the torch constricts the plasma and prevents the ICP from expanding to fill the outer tube, keeping the torch from melting. An RF induction coil, also called the load coil, surrounds the torch and produces an oscillating magnetic field, which in turn sets up an oscillating current in the ions and electrons produced from the argon. The impedance-matching unit serves to efficiently couple the RF energy from the generator to the load coil. The unit can be of either the active or the passive type. An active matching unit adjusts the impedance of the RF power by means of a capacitive network, whereas the passive type adjusts the impedance directly through the generator circuitry. Within the load coil of the RF generator, the energy transfer between the coil and the argon creates a self-sustaining plasma. Collisions of the ions and electrons liberated from the argon ionize and excite the analyte atoms in the high-temperature plasma. The plasma operates at temperatures of 6,000 to 10,000 K, so most covalent bonds and analyte-to-analyte interactions have been eliminated.

An inductively coupled plasma can use either an optical or a mass spectral detection system. In the former case, ICP–AES, analyte detection is achieved at an emission wavelength of the analyte in question. Because of differences in technology, a wide variety of ICP–AES systems are available, each with different capabilities, as well as different advantages and disadvantages. Simultaneous-detection systems are capable of analyzing multiple elements at the same time, thereby shortening analysis time and improving background detection and correction. Sequential systems move from one wavelength to the next to perform analyses, and often provide a larger number of analytical lines from which to choose. Array detectors, including charge-coupled devices and charge-injection devices, with detectors on a chip, make it possible to combine the advantages of both simultaneous and sequential systems. These types of detection devices are used in the most powerful spectrometers, providing rapid analysis and a wide selection of analytical lines.

The ICP can be viewed in either axial or radial (also called lateral) mode. The torch is usually positioned horizontally in axially viewed plasmas and is viewed end on, whereas it is positioned vertically in radially viewed plasmas and is viewed from the side. Axial viewing of the plasma can provide higher signal-to-noise ratios (better detection limits and precision); however, it also incurs greater matrix and spectral interferences. Methods validated on an instrument with a radial configuration will probably not be completely transferable to an instrument with an axial configuration, and vice versa.

Additionally, dual-view instrument systems are available, making it possible for the analyst to take advantage of either torch configuration. The selection of the optimal torch configuration will depend on the sample matrix, analyte in question, analytical wavelength(s) used, cost of instrumentation, required sensitivity, and type of instrumentation available in a given laboratory.

Regardless of torch configuration or detector technology, ICP–AES is a technique that provides a qualitative and/or quantitative measurement of the optical emission from excited atoms or ions at specific wavelengths. These measurements are then used to determine the analyte concentration in a given sample. Upon excitation, an atom or atomic ion emits an array of different frequencies of light that are characteristic of the distinct energy transition allowed for that element. The intensity of the light is generally proportional to the analyte concentration. It is necessary to correct for the background emission from the plasma. Sample concentration measurements are usually determined from a working curve of known standards over the concentration range of interest. It is, however, also possible to perform a single-point calibration under certain circumstances, such as with limit tests, if the methodology has been validated for sufficient specificity, sensitivity, linearity, accuracy, precision, ruggedness, and robustness.

Because there are distinct transitions between atomic energy levels, and because the atoms in an ICP are rather dilute, emission lines have narrow bandwidths. However, because the emission spectra from the ICP contain many lines, and because “wings” of these lines overlap to produce a nearly continuous background on top of the continuum that arises from the recombination of argon ions with electrons, a high-resolution spectrometer is required in ICP–AES. The decision regarding which spectral line to measure should include an evaluation of potential spectral interferences. All atoms in a sample are excited simultaneously; however, the presence of multiple elements in some samples can lead to spectral overlap. Spectral interference can also be caused by background emission from the sample or plasma. Modern ICPs usually have background correction available, and a number of background correction techniques can be applied. Simple background correction typically involves measuring the background emission intensity at some point away from the main peak and subtracting this value from the total signal being measured. Mathematical modeling to subtract the interfering signal as a background correction can also be performed with certain types of ICP–AES spectrometers.

The selection of the analytical spectral line is critical to the success of an ICP–AES analysis, regardless of torch configuration or detector type. Though some wavelengths are preferred, the final choice must be made in the context of the sample matrix, the type of instrument being used, and the sensitivity required. Analysts might choose to start with the wavelengths recommended by the manufacturer of their particular instrument and select alternative wavelengths based on manufacturer recommendations or published wavelength tables.3,4,5,6,7 Ultimately, the selection of analytical wavelengths should be demonstrated experimentally to provide sufficient specificity, sensitivity, linearity, accuracy, and precision of the analysis at hand.

Forward power, gas flow rates, viewing height, and torch position can all be optimized to provide the best signal. However, it must also be kept in mind that these same variables can influence matrix and spectral interferences.

In general, it is desirable to operate the ICP under robust conditions, which can be gauged on the basis of the MgII / MgI line pair at (280.270 nm / 285.213 nm). If that ratio of intensities is above 6.0 in an aqueous solution, the ICP is said to be robust, and is less susceptible to matrix interferences. A ratio of about 10.0 is generally what is sought. Note that the term robust conditions is unrelated to robustness as applied to analytical method validation. Operation of an instrument with an MgII / MgI ratio greater than 6.0 is not mandated, but is being suggested as a means of optimizing instrument parameters in many circumstances.

The analysis of the Group I elements can be an exception to this strategy. When atomic ions are formed from elements in this group, they assume a noble gas electron configuration, with correspondingly high excitation energy. Because the first excited state of these ions is extremely high, few are excited, so emission intensity is correspondingly low. This situation can be improved by reducing the fractional ionization, which can in turn be achieved by using lower forward power settings in combination with adjusted viewing height or nebulizer gas flow, or by adding an ionization suppression agent to the samples and standards.

When organic solvents are used, it is often necessary to use a higher forward power setting, higher intermediate and outer gas flows, and a lower nebulizer gas flow than would be employed for aqueous solutions, as well as a reduction in the nebulizer gas flow. When using organic solvents, it may also be necessary to bleed small amounts of oxygen into the torch to prevent carbon buildup in the torch.

Calibration

Standardization

Procedure

When an inductively coupled plasma uses a mass spectral detection system, the technique is referred to as inductively coupled plasma–mass spectrometry (ICP–MS). In this technique, analytes are detected directly at their atomic masses. Because these masses must be charged to be detected in ICP–MS, the method relies on the ability of the plasma source to both atomize and ionize sample constituents. As is the case with ICP–AES, a wide variety of ICP–MS instrumentation systems are available.

The systems most commonly in use are quadrupole-based systems. Gaining in interest is time-of-flight ICP–MS. Although still not in widespread use, this approach may see greater use in the future. Additionally, high-resolution sector field instruments are available.

Regardless of instrument design or configuration, ICP–MS provides both a qualitative and a quantitative measurement of the components of the sample. Ions are generated from the analyte atoms by the plasma. The analyte ions are then extracted from the atmospheric-pressure plasma through a sampling cone into a lower-pressure zone, ordinarily held at a pressure near 1 Torr. In this extraction process, the sampled plasma gases, including the analyte species, form a supersonic beam, which dictates many of the properties of the resulting analyte ions. A skimmer cone, located behind the sampling cone, “skims” the supersonic beam of ions as they emerge from the sampling cone. Behind the skimmer cone is a lower-pressure zone, often held near a milliTorr. Lastly, the skimmed ions pass a third-stage orifice to enter a zone held near a microTorr, where they encounter ion optics and are passed into the mass spectrometer. The mass spectrometer separates the ions according to their mass-to-charge (m/z) ratios. The ICP–MS has a mass range up to 240 atomic mass units (amu). Depending on the equipment configuration, analyte adducts can form with diluents, with argon, or with their decomposition products. Also formed are oxides and multiply-charged analyte ions, which can increase the complexity of the resulting mass spectra. Interferences can be minimized by appropriate optimization of operational parameters, including gas flows (central, intermediate, and outer gas flow rates), sample-solution flow, RF power, extraction-lens voltage, etc., or by the use of collision or reaction cells, or cool plasma operation, if available on a given instrument. Unless a laboratory is generating or examining isotopes that do not naturally occur, a list of naturally occurring isotopes will provide the analyst with acceptable isotopes for analytical purposes. Isotopic patterns also serve as an aid to element identification and confirmation. Additionally, tables of commonly found interferences and polyatomic isobaric interferences and correction factors can be used.

ICP–MS generally offers considerably lower (better) detection limits than ICP–AES, largely because of the extremely low background that it generates. This ability is a major advantage of ICP–MS for determination of very low analyte concentrations or when elimination of matrix interferences is required. In the latter case, some interferences can be avoided simply by additional dilution of the sample solution. In some applications, analytes can be detected below the parts per trillion (ppt) level using ICP–MS. As a general rule, ICP–MS as a technique requires that samples contain significantly less total dissolved solids than does ICP–AES.

The selection of the analytical mass to use is critical to the success of an ICP–MS analysis, regardless of instrument design. Though some masses are often considered to be the primary ones, because of their high natural abundance, an alternative mass for a given element is often used to avoid spectral overlaps (isobaric interferences). Selection of an analytical mass must always be considered in the context of the sample matrix, the type of instrument being used, and the concentrations to be measured. Analysts might choose to start with masses recommended by the manufacturer of their particular instrument and select alternate masses based on manufacturer's recommendations or published tables of naturally occurring isotopes.8

Optimization of an ICP–MS method is also highly dependent on the plasma parameters and means of sample introduction. Forward power, gas flow rates, and torch position may all be optimized to provide the best signal. When organic solvents are used, it is often necessary to use a higher forward power setting and a lower nebulizer flow rate than would be used for aqueous solutions. Additionally, when organic solvents are used, it might be necessary to introduce small amounts of oxygen into the central or intermediate gas to prevent carbon buildup in the torch or on the sampler cone orifice. The use of a platinum-tipped sampling or skimmer cone may also be required in order to reduce cone degradation with some organic solvents.

Calibration

Standardization

Procedure