Page 1196 - Veterinary Toxicology, Basic and Clinical Principles, 3rd Edition

1128 SECTION | XVII Analytical Toxicology

VetBooks.ir their atomic mass. Again, this provides for a very selec- with the same relative abundance levels. The standardiza-
tion of the electron energy at 70 eV for all instruments
tive (as well as a very sensitive) method of analysis.
provides consistency in mass spectra among instruments
A mass spectrometer consists of three essential
components: an ion source, a mass analyzer, and a from different manufacturers and different laboratories.
detector. The mass analyzer and detector (and in some Therefore, a compound analyzed in one laboratory will
cases, the ion source) are maintained under high provide the same spectrum when it’s analyzed in any other
vacuum, so a vacuum system of some kind is also laboratory. This has allowed for the development of mass
important. While a wide variety of ion sources and spectral libraries, containing spectra for hundreds of thou-
mass analyzers have been developed, only a few of sands of different chemicals. Instrument software is pro-
these are commonly used in veterinary diagnostics. vided with most GC-MS systems, which allows the
These are described below. analyst to search an obtained mass spectrum against all of
those contained within the library. This library searching
Ion Sources software provides an excellent means of identifying both
targeted compounds suspected to be present and non-
For a compound to be analyzed by mass spectrometry, it
targeted ones that are not expected to be present in a sam-
must be present in the form of an ion; i.e., it must have a
ple at the time of analysis.
positive or negative charge on it. The vast majority of
compounds of interest in veterinary diagnostics are not
naturally present as ions. Therefore, some method of ion-
Electrospray Ionization (ESI)
izing them must be used in order for them to be analyzed
Electrospray is the ionization method typically used in
by MS. Common ionization methods used in veterinary
HPLC-MS. The coupling of GC with MS was a relatively
diagnostics include ICP for elements, electron ionization
easy undertaking as compared to coupling HPLC to MS.
(EI) for GC-MS, and electrospray for LC-MS.
This is primarily due to the fact that mass analyzers must
operate in a vacuum. Ions formed in an ion source must be
ICP
physically moved to the mass analyzer, and this cannot
In ICP-MS, the ICP torch functions as the ion source for
occur if there are significant numbers of other molecules
the instrument. The ionized elements are then directed to
such as atmospheric nitrogen, oxygen, or water that get in
the mass analyzer where they are detected based on their
the way. A typical GC system will introduce somewhere
m/z ratios.
around 1 cc of carrier gas per minute into the mass spec-
trometer, and this amount of gas is easily pumped away
Electron Ionization (EI) using common types of vacuum pumps. A liquid chro-
This is the technique commonly used in GC-MS. In EI, matograph eluting directly into a mass spectrometer would
the ion source is housed in the vacuum chamber of the introduce somewhere between 0.1 and 0.5 mL/min of sol-
mass spectrometer. An electrical current is applied to a fil- vent into the MS. This volume of solvent expands tremen-
ament consisting of a thin wire. The current is set such dously when vaporized, to the point that only extremely
that electrons are emitted from the filament with an energy complex and expensive vacuum systems could pump it
of 70 eV. The end of the GC column is placed such that away and maintain vacuum. Various techniques were used
compounds elute from the column directly into the stream to try to get around this issue, but none were seen as prac-
of electrons emitted from the filament (Fig. 81.8). A com- tical until the development of electrospray ionization. This
pound impacted by these electrons will emit an electron of technique involves directing the LC flow through a fine
its own, resulting in a positive charge on the compound. diameter needle. Voltage is applied to the needle as the
Additionally, the high energy level of these filament elec- solvent flows through it, resulting in a fine spray of
trons will cause many compounds to break apart, with charged droplets. (This is somewhat similar to how an
some of the fragments being ionized while others remain electric paint sprayer works.) The droplets are directed at
neutral. The resulting ions (intact molecules and/or their the front of the mass spectrometer. The electric charge
fragments) are then directed into the mass analyzer, which helps to ionize molecules within the droplets while solvent
differentiates them based on m/z. The output of this pro- from the HPLC mobile phase evaporates from the droplets
cess is what is typically recognized as a mass spectral as they pass between the needle and the mass spectrome-
“fingerprint,” showing the m/z and relative abundance of ter. In this manner, the bulk of the liquid is eliminated
each of the ions. Note that in Fig. 81.8, each of the signals while any analytes present are simultaneously ionized
represents a different fragment of the caffeine molecule. (Fig. 81.9). This ionization mechanism differs significantly
Keeping the electron energy precisely set at 70 eV ensures from EI, providing intact molecular ions without the frag-
that the fragmentation of a given compound will happen mentation of EI. So instead of a somewhat unique molecu-
consistently, always providing the same fragment ions lar fingerprint, ESI usually produces ions of a single m/z