Samples are introduced either as a gas to be ionized in the ion source, or by ejection of charged molecular species from a solid surface or solution. In some cases sample introduction and ionization take place in a single process, making a distinction between them somewhat artificial.

Substances that are gases or liquids at room temperature and atmospheric pressure can be admitted to the source as a neutral beam via a controllable leak system. Volatilizable compounds dissolved or adsorbed in solids or liquids can be removed and concentrated with a headspace analyzer. Vapors are flushed from the solid or liquid matrix with a stream of carrier gas and trapped on an adsorbing column. The trapped vapors are subsequently desorbed by programmed heating of the trap and introduced into the mass spectrometer by a capillary connection.

For volatilizable solids, the most frequently used method of sample introduction is the direct insertion probe. Here, the sample is placed in a small crucible at the tip of the probe, which is heated under high vacuum in close proximity to the ion source. A variation of this technique involves desorption of samples inside the ionization chamber from a rapidly heated wire or with the aid of a laser beam. Such desorption techniques, in combination with electron, chemical, or field ionization, are preferred for the analysis of heat sensitive or poorly volatile samples.

Sample introduction techniques that involve the ejection of charged molecules from the surface of solid samples include the field desorption method and various sputtering techniques, where the samples are bombarded by high energy photons, by a primary ion beam, or by a neutral particle beam. Similarly, ions can be ejected from solutions either by bombardment with a primary beam, or by one of the various spray techniques described below.

Gas and liquid chromatographs are widely used as sample inlet devices for mass spectrometers. These chromatographs provide for an initial sample purification, since only that portion of the chromatographic effluent containing the compound of interest need be admitted to the mass spectrometer. Gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) combinations are valuable tools for the identification of unknown impurities in drugs. These combination methods have the capacity to separate complex mixtures with the opportunity to obtain structural information on the individual components.

Gas Chromatography/Mass Spectrometry

Liquid Chromatography/Mass Spectrometry

Electron Impact

Chemical Ionization (CI)

Fast Atom Bombardment (FAB)

Mass analyzers separate the charged species in the ionized sample according to their m/z ratios, thus permitting the mass and abundance of each species to be determined. Four commonly used analyzers are the magnetic sector, the quadrupole, the time-of-flight, and the Fourier transform analyzers.

Magnetic Sector Analyzers

Quadrupole Analyzers

Ion-trap Analyzer

Time-of-flight Analyzers

Fourier Transform Analyzers

Two mass spectrometers connected in series (MS/MS), tandem mass spectrometry, refers to the use of two or more sequential mass analysis steps. In its simplest form MS/MS (Figure 2)

Fig. 2. Tandem Mass Spectrometry.

consists of two mass spectrometers linked in such a way that ions preselected by the first mass analyzer (MS1) are chemically or energetically modified and the results analyzed by the second mass analyzer (MS2).

The basic concept of MS/MS involves the ability to determine the mass relationship between a precursor ion in MS1 and a product ion in MS2. Different mass relationships can be probed depending on how MS1 and MS2 are scanned. These include fragmentation of a precursor and measurement of all its fragments (a product scan), selection of multiple precursors and testing for a common fragment (a precursor scan), or scanning to see if a number of precursors all lose the same neutral species (a constant neutral loss scan).

Fragmentation of the precursor ion can be induced by momentum transfer through collision with gas molecules and/or solid surfaces or by electronic excitation using lasers. These techniques are known as collision-induced dissociation, surface-induced dissociation, or laser-induced dissociation, respectively. Allowing the ion to fragment without additional activation is known as metastable decomposition.

There are many applications of MS/MS to pharmaceutical problems. Product scans can be used to obtain qualitative information from precursor ions of drug substances, impurities, and contaminants. This can aid in the identification of unknowns. The method can also be used to determine the amino acid sequence of peptides and protein fragments.

MS/MS has advantages for mixture analysis. Even when the mass spectrometer is coupled to a separation device such as a liquid or gas chromatograph, the resulting signals may be a result of overlapping or unresolved components. MS/MS can be employed to select the precursor ion from one component and obtain structural information without interference from the others.

Selected reaction monitoring is used to reduce the interference encountered during quantitative analysis for low levels of drugs in biological matrices, as in pharmacokinetic studies. If analysis is for a drug specific ion, interfering signals from other compounds in the matrix can mask the desired signal. Interference is reduced if a drug-specific fragment is selected with MS1 and a structure-specific fragment with MS2. The odds of another molecule producing the same mass relationship are diminishingly small.

MS/MS can also be used in metabolism studies to search for molecules with common structural features such as metabolites related to the drug substance. All of the metabolites might contain the same functional group that is lost as a neutral fragment. In this case a contant-neutral-loss scan will show all of these species. For instance, carboxylic acids will all lose neutral carbon dioxide. If the common functionality is lost as an ionic fragment, then a precursor scan will show all of the molecules that produce that fragment ion.

The mass spectral experiment yields information on the molecular weight of ions derived from the sample and the relative abundance of each of these ions. Spectra are often complex, and not all of the ions may be separated by the mass spectrometer. The ability of the instrument to separate ions is called the resolving power, commonly described by the “10% valley” definition. This states that the resolving power is the highest mass number at which two peaks differing by one molecular weight unit and of equal height have a valley between them that is equal to 10% of the peak height. For low, medium, and high resolution mass spectrometers, this value is between 100 and 2000, 2000 and 10,000, and greater than 10,000, respectively.

If one electron is removed or added to a neutral molecule, a molecular ion of essentially the same molecular weight as the parent molecule results. It is often possible to determine the mass of this ion with sufficient precision to enable the empirical formula of the compound to be calculated. Molecular masses may be determined accurately by using high resolution instruments or by peak-matching measurements using reference compounds.

Fragment ions are those produced from the molecular ion by various bond cleavage processes. Numerous papers in the literature relate bond cleavage patterns (fragmentation patterns) to molecular structure.

In addition to measurement of the mass of a molecular ion and its associated fragment ions, mass spectrometers are also used to quantitate compounds with a high degree of selectivity, precision, and accuracy. Compounds are introduced into the mass spectrometer either via direct insertion probe, gas inlet, or, as is more common, via gas or liquid chromatographic interfaces, which provide sample purification. Ionization may be by EI, CI, FAB, thermospray, or electrospray and mass separation by magnetic sector, quadrupole, or quadrupole ion-trap mass spectrometers. Quantitative mass spectrometry involves measuring the abundance of a specific ion, or set of ions, and relating the response to a known standard. External or internal standards may be used, but the latter are preferred for greater accuracy.

For mass spectrometry, internal standards may be either structural or stable isotope analogs. The former have the advantage of lower cost and availability while precision and accuracy are typically achieved by use of a stable isotope (2H, 13C, 15N) labeled analog of the analyte. The only requirements for labeling the analyte are that the ion monitored for the internal standard must retain an isotopic label after ionization and the label must not be exchangeable under the sampling, separation, or ionization conditions. Stable isotope internal standards are often required for acceptable quantitation, particularly with FAB and LC/MS techniques such as thermospray and electrospray.

Relative abundances of the analyte and internal standard ions are typically determined by selected ion monitoring, by which only specific ions due to the analyte and the internal standard are monitored. This technique has the advantage over scanning the full mass range in that more time is spent integrating the ion current at a selected mass-to-charge ratio, thereby increasing sensitivity. Chromatographic peak area or amount of analyte in a sample is calculated from the ratio of analyte to internal standard peak area (or height) and the regression parameters as determined by a calibration curve, using standard techniques.