Appendix II G. Mass Spectrometry

Mass spectrometry is based on the direct measurement of the ratio of the mass to the number of positive or negative elementary charges of ions (m/z) in the gas phase obtained from the substance to be analysed. This ratio is expressed in atomic mass units (1 a.m.u. = one twelfth the mass of ¹²C) or in daltons (1 Da = the mass of the hydrogen atom).

The ions, produced in the ion source of the apparatus, are accelerated and then separated by the analyser before reaching the detector. All of these operations take place in a chamber where a pumping system maintains a vacuum of 10^-3 to 10^-6 Pa.

The resulting spectrum shows the relative abundance of the various ionic species present as a function of m/z. The signal corresponding to an ion will be represented by several peaks corresponding to the statistical distribution of the various isotopes of that ion. This pattern is called the isotopic profile and (at least for small molecules) the peak representing the most abundant isotopes for each atom is called the monoisotopic peak.

Information obtained in mass spectrometry is essentially qualitative (determination of the molecular mass, information on the structure from the fragments observed) or quantitative (using internal or external standards) with limits of detection ranging from the picomole to the femtomole.

The very first step of an analysis is the introduction of the sample into the apparatus without overly disturbing the vacuum. In a common method, called direct liquid introduction, the sample is placed on the end of a cylindrical rod (in a quartz crucible, on a filament or on a metal surface). This rod is introduced into the spectrometer after passing through a vacuum lock where a primary intermediate vacuum is maintained between atmospheric pressure and the secondary vacuum of the apparatus.

Other introduction systems allow the components of a mixture to be analysed as they are separated by an appropriate apparatus connected to the mass spectrometer.

Gas chromatography/mass spectrometry The use of suitable columns (capillary or semi-capillary) allows the end of the column to be introduced directly into the source of the apparatus without using a separator.

Liquid chromatography/mass spectrometry This combination is particularly useful for the analysis of polar compounds, which are insufficiently volatile or too heat-labile to be analysed by gas chromatography coupled with mass spectrometry. This method is complicated by the difficulty of obtaining ions in the gas phase from a liquid phase, which requires very special interfaces such as:

• — direct liquid introduction: the mobile phase is nebulised, and the solvent is evaporated in front of the ion source of the apparatus,

• — particle-beam interface: the mobile phase, which may flow at a rate of up to 0.6 mL/min, is nebulised in a desolvation chamber such that only the analytes, in neutral form, reach the ion source of the apparatus; this technique is used for compounds of relatively low polarity with molecular masses of less than 1000 Da,

• — moving-belt interface: the mobile phase, which may flow at a rate of up to 1 mL/min, is applied to the surface of a moving belt; after the solvent evaporates, the components to be analysed are successively carried to the ion source of the apparatus where they are ionised; this technique is rather poorly suited to very polar or heat-labile compounds.

Other types of coupling (electrospray, thermospray, atmospheric-pressure chemical ionisation) are considered to be ionisation techniques in their own right and are described in the section on modes of ionisation.

Supercritical fluid chromatography/mass spectrometry The mobile phase, usually consisting of supercritical carbon dioxide enters the gas state after passing a heated restrictor between the column and the ion source.

Capillary electrophoresis/mass spectrometry The eluent is introduced into the ion source, in some cases after adding another solvent so that flow rates of the order of a few microlitres per minute can be attained. This technique is limited by the small quantities of sample introduced and the need to use volatile buffers.

Electron impact The sample, in the gas state, is ionised by a beam of electrons whose energy (usually 70 eV) is greater than the ionisation energy of the sample. In addition to the molecular ion M⁺, fragments characteristic of the molecular structure are observed. This technique is limited mainly by the need to vaporise the sample. This makes it unsuited to polar, heat-labile or high molecular mass compounds. Electron impact is compatible with the coupling of gas chromatography to mass spectrometry and sometimes with the use of liquid chromatography.

Chemical ionisation This type of ionisation involves a reagent gas such as methane, ammonia, nitrogen oxide, nitrogen dioxide or oxygen. The spectrum is characterised by ions of the (M + H)⁺ or (M - H)^– types, or adduct ions formed from the analyte and the gas used. Fewer fragments are produced than with electron impact. A variant of this technique is used when the substance is heat-labile: the sample, applied to a filament, is very rapidly vaporised by the Joule-Thomson effect (desorption chemical ionisation).

Fast-atom bombardment (FAB) or fast-ion bombardment ionisation (liquid secondary-ion mass spectrometry LSIMS) The sample, dissolved in a viscous matrix such as glycerol, is applied to a metal surface and ionised by a beam of neutral atoms such as argon or xenon or high-kinetic-energy caesium ions. Ions of the (M + H)⁺ or (M - H)^– types or adduct ions formed from the matrix or the sample are produced. This type of ionisation, well suited to polar and heat-labile compounds, allows molecular masses of up to 10 000 Da to be obtained. The technique can be combined with liquid chromatography by adding 1 per cent to 2 per cent of glycerol to the mobile phase; however, the flow rates must be very low (a few microlitres per minute). These ionisation techniques also allow thin-layer chromatography plates to be analysed by applying a thin layer of matrix to the surface of these plates.

Field desorption and field ionisation and field ionisation The sample is vaporised near a tungsten filament covered with microneedles (field ionisation) or applied to this filament (field desorption). A voltage of about 10 kV, applied between this filament and a counter-electrode, ionises the sample. These two techniques mainly produce molecular ions M⁺, and (M + H)⁺ ions and are used for low polarity and/or heat-labile compounds.

Matrix-assisted laser desorption ionisation (MALDI) The sample, in a suitable matrix and deposited on a metal support, is ionised by a pulsed laser beam whose wavelength may range from UV to IR (impulses lasting from a picosecond to a few nanoseconds). This mode of ionisation plays an essential role in the analysis of very high molecular mass compounds (more than 100 000 Da) but is limited to time-of flight analysers (see below).

Electrospray This mode of ionisation is carried out at atmospheric pressure. The samples, in solution, are introduced into the source through a capillary tube, the end of which has a potential of the order of 5 kV. A gas can be used to facilitate nebulisation. Desolvation of the resulting microdroplets produces singly or multiply charged ions in the gas phase. The flow rates vary from a few microlitres per minute to 1 mL/min. This technique is suited to polar compounds and to the investigation of biomolecules with molecular masses of up to 100 000 Da. It can be coupled to liquid chromatography or capillary electrophoresis.

Atmospheric-pressure chemical ionisation (APCI) Ionisation is carried out at atmospheric pressure by the action of an electrode maintained at a potential of several kilovolts and placed in the path of the mobile phase, which is nebulised both by thermal effects and by the use of a stream of nitrogen. The resulting ions carry a single charge and are of the (M + H)⁺ type in the positive mode and of the (M - H)^– type in the negative mode. The high flow rates that can be used with this mode of ionisation (up to 2 mL/min) make this an ideal technique for coupling to liquid chromatography.

Thermospray The sample, in the mobile phase consisting of water and organic modifiers and containing a volatile electrolyte (generally ammonium acetate) is introduced in nebulised form after having passed through a metal capillary tube at controlled temperature. Acceptable flow rates are of the order of 1 mL/min to 2 mL/min. The ions of the electrolyte ionise the compounds to be analysed. This ionisation process may be replaced or enhanced by an electrical discharge of about 800 volts, notably when the solvents are entirely organic. This technique is compatible with the use of liquid chromatography coupled with mass spectrometry.

Differences in the performance of analysers depend mainly on two parameters:

• — the range over which m/z ratios can be measured, ie, the mass range,

• — their resolving power characterised by the ability to separate two ions of equal intensity with m/z ratios differing by ΔM, and whose overlap is expressed as a given percentage of valley definition; for example, a resolving power (M/ΔM) of 1000 with 10 per cent valley definition allows the separation of m/z ratios of 1000 and 1001 with the intensity returning to 10 per cent above baseline. However, the resolving power may in some cases (time-of-flight analysers, quadrupoles, ion-trap analysers) be defined as the ratio between the molecular mass and peak width at half height (50 per cent valley definition).

Magnetic and electrostatic analysers The ions produced in the ion source are accelerated by a voltage V, and focused towards a magnetic analyser (magnetic field B) or an electrostatic analyser (electrostatic field E), depending on the configuration of the instrument. They follow a trajectory of radius r according to Laplace's law:

Two types of scans can be used to collect and measure the various ions produced by the ion source: a scan of B holding V fixed or a scan of V with constant B. The magnetic analyser is usually followed by an electric sector that acts as a kinetic energy filter and allows the resolving power of the instrument to be increased appreciably. The maximum resolving power of such an instrument (double sector) ranges from 10 000 to 150 000 and in most cases allows the value of m/z ratios to be calculated accurately enough to determine the elemental composition of the corresponding ions. For monocharged ions, the mass range is from 2000 Da to 15 000 Da. Some ions may decompose spontaneously (metastable transitions) or by colliding with a gas (collision-activated dissociation (CAD)) in field-free regions between the ion source and the detector. Examination of these decompositions is very useful for the determination of the structure as well as the characterisation of a specific compound in a mixture and involves tandem mass spectrometry. There are many such techniques depending on the region where these decompositions occur:

• — daughter-ion mode (determination of the decomposition ions of a given parent ion): B/E = constant, MIKES (Mass-analysed Ion Kinetic Energy Spectroscopy),

• — parent-ion mode (determination of all ions which by decomposition give an ion with a specific m/z ratio): B²/E = constant,

• — neutral-loss mode (detection of all the ions that lose the same fragment):

• B/E(1 - E/E₀)^1/2 = constant, where E₀ is the basic voltage of the electric sector.

Quadrupoles The analyser consists of four parallel metal rods, which are cylindrical or hyperbolic in cross-section. They are arranged symmetrically with respect to the trajectory of the ions; the pairs diagonally opposed about the axis of symmetry of rods are connected electrically. The potentials to the two pairs of rods are opposed. They are the resultant of a constant component and an alternating component. The ions produced at the ion source are transmitted and separated by varying the voltages applied to the rods so that the ratio of continuous voltage to alternating voltage remains constant. The quadrupoles usually have a mass range of 1 a.m.u. to 2000 a.m.u., but some may range up to 4000 a.m.u. Although they have a lower resolving power than magnetic sector analysers, they nevertheless allow the monoisotopic profile of single charged ions to be obtained for the entire mass range. It is possible to obtain spectra using three quadrupoles arranged in series, Q₁, Q₂, Q₃ (Q₂ serves as a collision cell and is not really an analyser; the most commonly used collision gas is argon).

The most common types of scans are the following:

• — daughter-ion mode: Q₁ selects an m/z ion whose fragments obtained by collision in Q₂ are analysed by Q₃,

• — parent-ion mode: Q₃ filters only a specific m/z ratio, while Q₁ scans a given mass range. Only the ions decomposing to give the ion selected by Q₃ are detected,

• — neutral loss mode: Q₁ and Q₃ scan a certain mass range but at an offset corresponding to the loss of a fragment characteristic of a product or family of compounds.

It is also possible to obtain spectra by combining quadrupole analysers with magnetic or electrostatic sector instruments; such instruments are called hybrid mass spectrometers.

Ion-trap analyser The principle is the same as for a quadrupole, this time with the electric fields in three dimensions. This type of analyser allows product-ion spectra over several generations (MSⁿ) to be obtained.

Ion-cyclotron resonance analysers Ions produced in a cell and subjected to a uniform, intense magnetic field move in circular orbits at frequencies which can be directly correlated to their m/z ratio by applying a Fourier transform algorithm. This phenomenon is called ion-cyclotron resonance. Analysers of this type consist of superconducting magnets and are capable of very high resolving power (up to 1000 000 and more) as well as MSⁿ spectra. However, very low pressures are required (of the order of 10^-7 Pa).

Time-of-flight analysers The ions produced at the ion source are accelerated at a voltage V of 10 kV to 20 kV. They pass through the analyser, consisting of a field-free tube, 25 cm to 1.5 m long, generally called a flight tube. The time (t) for an ion to travel to the detector is proportional to the square root of the m/z ratio. Theoretically the mass range of such an analyser is infinite. In practice, it is limited by the ionisation or desorption method. Time-of-flight analysers are mainly used for high molecular mass compounds (up to several hundred thousand daltons). This technique is very sensitive (a few picomoles of product are sufficient). The accuracy of the measurements and the resolving power of such instruments may be improved considerably by using an electrostatic mirror (reflectron).

There are essentially three possible modes.

Complete spectrum mode The entire signal obtained over a chosen mass range is recorded. The spectrum represents the relative intensity of the different ionic species present as a function of m/z. The results are essentially qualitative. The use of spectral reference libraries for more rapid identification is possible.

Fragmentometric mode (Selected-ion monitoring) The acquired signal is limited to one (single-ion monitoring (SIM)) or several (multiple-ion monitoring (MIM)) ions characteristic of the substance to be analysed. The limit of detection can be considerably reduced in this mode. Quantitative or semiquantitative tests can be carried out using external or internal standards (for example, deuterated standards). Such tests cannot be carried out with time-of-flight analysers.

Fragmentometric double mass spectrometry mode (multiple reaction monitoring (MRM)) The unimolecular or bimolecular decomposition of a chosen precursor ion characteristic of the substance to be analysed is followed specifically. The selectivity and the highly specific nature of this mode of acquisition provide excellent sensitivity levels and make it the most appropriate for quantitative studies using suitable internal standards (for example, deuterated standards). This type of analysis can be performed only on apparatus fitted with three quadrupoles in series, ion-trap analysers or cyclotron-resonance analysers.

Calibration allows the corresponding m/z value to be attributed to the detected signal. As a general rule, this is done using a reference substance. This calibration may be external (acquisition file separate from the analysis) or internal (the reference substance(s) are mixed with the substance to be examined and appear on the same acquisition file). The number of ions or points required for reliable calibration depends on the type of analyser and on the desired accuracy of the measurement, for example, in the case of a magnetic analyser where the m/z ratio varies exponentially with the value of the magnetic field, there should be as many points as possible.

Ions separated by an analyser are converted into electric signals by a detection system such as a photomultiplier or an electron multiplier. These signals are amplified before being re-converted into digital signals for data processing, allowing various functions such as calibration, reconstruction of spectra, automatic quantification, archiving, creation or use of libraries of mass spectra. The various physical parameters required for the functioning of the apparatus as a whole are controlled by computer.

Inductively coupled plasma-mass spectrometry (ICP-MS) is a mass spectrometry method that uses an inductively coupled plasma (ICP) as the ionisation source. The basic principles of ICP formation are described in chapter 2.2.57 on inductively coupled plasma-atomic emission spectrometry (ICP-AES).

ICP-MS utilises the ability of the ICP to generate charged ions from the element species within a sample. These ions are then directed into a mass spectrometer, which separates them according to their mass-to-charge ratio (m/z). Most mass spectrometers have a quadrupole system or a magnetic sector. Ions are transported from the plasma through 2 cones (sampler and skimmer cones, forming the interface region) to the ion optics. The ion optics consist of an electrostatic lens, which takes ions from an area at atmospheric pressure to the mass filter at a vacuum of 10^-8 Pa or less, maintained with a turbomolecular pump. After their filtration, ions of the selected mass/charge ratio are directed to a detector (channel electromultiplier, Faraday cup, dynodes), where ion currents are converted into electrical signals. The element is quantified according to the number of ions arriving and generating electrical pulses per unit time.

The sample-introduction system and data-handling techniques of an ICP-AES system are also used in ICP-MS.

The apparatus consists essentially of the following elements:

• — sample-introduction system, consisting of a peristaltic pump delivering the solution at constant flow rate into a nebuliser;

• — radio-frequency (RF) generator;

• — plasma torch;

• — interface region including cones to transport ions to the ion optics;

• — mass spectrometer;

• — detector;

• — data-acquisition unit.

Mass interference is the major problem, for example by isobaric species that significantly overlap the mass signal of the ions of interest, especially in the central part of the mass range (for example 40-80 a.m.u.). The combination of atomic ions leads to polyatomic or molecular interferences (i.e. ⁴⁰Ar¹⁶O with ⁵⁶Fe or ⁴⁰Ar⁴⁰Ar with ⁸⁰Se). Matrix interference may also occur with some analytes. Some samples have an impact on droplet formation or on the ionisation temperature in the plasma. These phenomena may lead to the suppression of analyte signals. Physical interference is to be circumvented by using the method of internal standardisation or by standard addition. The element used as internal standard depends on the element to be measured: ⁵⁹Co and ¹¹⁵In, for example, can be used as internal standards.

The prime characteristic of an ICP-MS instrument is its resolution, i.e. the efficiency of separation of 2 close masses. Quadrupole instruments are, from this point of view, inferior to magnetic-sector spectrometers.

The sample preparation usually involves a step of digestion of the matrix by a suitable method, for example in a microwave oven. Furthermore, it is important to ensure that the analyte concentration falls within the working range of the instrument through dilution or preconcentration, and that the sample-containing solution can be nebulised in a reproducible manner.

Several sample-introduction systems tolerate high acid concentrations, but the use of sulfuric and phosphoric acids can contribute to background emission. Therefore, nitric and hydrochloric acids are preferable. The availability of hydrofluoric acid-resistant (for example perfluoroalkoxy polymer) sample-introduction systems and torches also allows the use of hydrofluoric acid. In selecting a sample-introduction method, the requirements for sensitivity, stability, speed, sample size, corrosion resistance and resistance to clogging have to be considered. The use of a cross-flow nebuliser combined with a spray chamber and torch is suitable for most requirements. The peristaltic pumps usually deliver the standard and sample solutions at a rate of 20-1000 µL/min.

In the case of organic solvents being used, the introduction of oxygen must be considered to avoid organic layers.

The standard operating conditions prescribed by the manufacturer are to be followed. Usually, different sets of operating conditions are used for aqueous solutions and for organic solvents. Suitable operating parameters are to be properly chosen:

• — selection of cones (material of sampler and skimmer);

• — support-gas flow rates (outer, intermediate and inner tubes of the torch);

• — RF power;

• — pump speed;

• — selection of one or more isotopes of the element to be measured (mass).

Isotope selection is made using several criteria. The most abundant isotope for a given element is selected to obtain maximum sensitivity. Furthermore, an isotope with the least interference from other species in the sample matrix and from the support gas should be selected. Information about isobaric interferences and interferences from polyatomic ions of various types, for example hydrides, oxides, chlorides, etc., is usually available in the software of ICP-MS instrument manufacturers.

• — Tuning of the instrument allows to monitor and adjust the measurement before running samples. ICP-MS mass accuracy is checked with a tuning solution containing several isotopes covering the whole range of masses, for example ⁹Be, ⁵⁹Co, ⁸⁹Y, ¹¹⁵In, ¹⁴⁰Ce and ²⁰⁹Bi.

• — Sensitivity and short- and long-term stability are recorded. The instrument parameters (plasma condition, ion lenses and quadrupole parameter) are to be optimised to obtain the highest possible number of counts.

• — Tuning for resolution and mass axis is to be done with a solution of Li, Y and Tl to ensure an acceptable response over a wide range of masses.

• — Evaluation of the efficiency of the plasma to decompose oxides has to be performed in order to minimise these interferences. The ratio Ce/CeO and/or Ba/BaO is a good indicator, and a level less than about 3 per cent is required.

• — Reduction of the formation of double-charged ions is made with Ba and Ce. The ratio of the signal for double-charged ions to the assigned element should be less than 2 per cent.

• — Long-term stability is checked by running a standard first and at the end of the sample sequence, controlling whether salt deposits on the cones have reduced the signal throughout the run.

Satisfactory performance of methods prescribed in monographs is verified at suitable time intervals.

Prepare and analyse not fewer than 4 reference solutions over the calibration range plus a blank. Perform not fewer than 5 replicates.

The calibration curve is calculated by least-square regression from all measured data of the calibration test. The regression curve, the means, the measured data and the confidence interval of the calibration curve are plotted. The operating method is valid when:

• — the correlation coefficient is at least 0.99;

• — the residuals of each calibration level are randomly distributed around the calibration curve.

Calculate the mean and relative standard deviation for the lowest and for the highest calibration level.

When the ratio of the estimated standard deviations of the lowest and the highest calibration level is less than 0.5 or greater than 2.0, a more precise estimation of the calibration curve may be obtained using weighted linear regression. Both linear and quadratic weighting functions are applied to the data to find the most appropriate weighting function to be employed.

If the means compared to the calibration curve show a deviation from linearity, two-dimensional linear regression is used.

Verify the accuracy preferably by using a certified reference material (CRM). Where this is not possible, perform a test for recovery.

Recovery For assay determinations a recovery of 90 per cent to 110 per cent is to be obtained. The test is not valid if recovery, for example for trace-element determination, is outside the range 80 per cent to 120 per cent of the theoretical value. Recovery may be determined on a suitable reference solution (matrix solution) spiked with a known quantity of analyte (concentration range that is relevant to the samples to be determined).

The repeatability is not greater than 3 per cent for an assay and not greater than 5 per cent for an impurity test.

Verify that the limit of quantification (for example, determined using the 10 σ approach) is below the value to be measured.