Liquid
chromatography-mass spectrometry (LC-MS) is now a routine technique with
the development of electrospray ionisation (ESI) providing a simple and
robust interface. It can be applied to a wide range of biological
molecules and the use of tandem MS and stable isotope internal standards
allows highly sensitive and accurate assays to be developed although
some method optimisation is required to minimise ion suppression
effects. Fast scanning speeds allow a high degree of multiplexing and
many compounds can be measured in a single analytical run. With the
development of more affordable and reliable instruments, LC-MS is
starting to play an important role in several areas of clinical
biochemistry and compete with conventional liquid chromatography and
other techniques such as immunoassay.
Introduction
Coupling
of MS to chromatographic techniques has always been desirable due to
the sensitive and highly specific nature of MS compared to other
chromatographic detectors. The coupling of gas chromatography to MS
(GC-MS) was achieved in the 1950s with commercial instruments available
from the 1970s. Relatively cheap and reliable GC-MS systems are now a
feature of many clinical biochemistry laboratories and are indispensable
in several areas where the analysis of complex mixtures and unambiguous
identification is required e.g. screening urine samples for inborn
errors of metabolism or drugs. The coupling of MS with LC (LC-MS) was an
obvious extension but progress in this area was limited for many years
due to the relative incompatibility of existing MS ion sources with a
continuous liquid stream. Several interfaces were developed but they
were cumbersome to use and unreliable, so uptake by clinical
laboratories was very limited. This situation changed with the
development of the electrospray ion source by Fenn in the 1980s.
The
following popper user interface control may not be accessible. Tab to
the next button to revert the control to an accessible version.
Destroy user interface control1
Manufacturers rapidly developed instruments equipped with electrospray
sources, which had a great impact on protein and peptide biochemistry.
Fenn was awarded the Nobel Prize in 2002 with Koichi Tanaka who
developed matrix assisted laser desorption ionisation, another extremely
useful MS ionisation technique for the analysis of biological
molecules.
The following
popper user interface control may not be accessible. Tab to the next
button to revert the control to an accessible version.
Destroy user interface control2
By
the mid 1990s, the price and performance of LC-MS instruments had
improved to the extent that clinical biochemistry laboratories were able
to take advantage of the new technology. Biochemical genetics was one
of the first areas to do so, and the analysis of neonatal dried blood
spot samples for a range of inborn errors of metabolism was a major
early application.
The
following popper user interface control may not be accessible. Tab to
the next button to revert the control to an accessible version.
Destroy user interface control3
There are a number of other clinical applications of LC-MS, and the
technique is more generally applicable than GC-MS owing to the broader
range of biological molecules that can be analysed and the greater use
of LC separations in clinical laboratories. The reasons for choosing
LC-MS over LC with conventional detectors are essentially the same as
with GC-MS, namely high specificity and the ability to handle complex
mixtures. Applications of electrospray MS were reviewed in The Clinical Biochemist Reviews in 2003.
The
following popper user interface control may not be accessible. Tab to
the next button to revert the control to an accessible version.
Destroy user interface control4
The current review focuses on the principles of LC-MS, practical
considerations in setting up LC-MS assays and reviews some of the major
applications in clinical biochemistry, concentrating on small molecule
applications.Mass Spectrometry Instrumentation
Mass
spectrometers operate by converting the analyte molecules to a charged
(ionised) state, with subsequent analysis of the ions and any fragment
ions that are produced during the ionisation process, on the basis of
their mass to charge ratio (m/z). Several different
technologies are available for both ionisation and ion analysis,
resulting in many different types of mass spectrometers with different
combinations of these two processes. In practice, some configurations
are far more versatile than others and the following descriptions focus
on the major types of ion sources and mass analysers likely to be used
in LC-MS systems within clinical laboratories.
Ion Sources
Electrospray Ionisation Source
Fenn
developed ESI into a robust ion source capable of interfacing to LC and
demonstrated its application to a number of important classes of
biological molecules.
The
following popper user interface control may not be accessible. Tab to
the next button to revert the control to an accessible version.
Destroy user interface control1
ESI works well with moderately polar molecules and is thus well suited
to the analysis of many metabolites, xenobiotics and peptides. Liquid
samples are pumped through a metal capillary maintained at 3 to 5 kV and
nebulised at the tip of the capillary to form a fine spray of charged
droplets. The capillary is usually orthogonal to, or off-axis from, the
entrance to the mass spectrometer in order to minimise contamination.
The droplets are rapidly evaporated by the application of heat and dry
nitrogen, and the residual electrical charge on the droplets is
transferred to the analytes.
The
following popper user interface control may not be accessible. Tab to
the next button to revert the control to an accessible version.
Destroy user interface control5
The ionised analytes are then transferred into the high vacuum of the
mass spectrometer via a series of small apertures and focusing voltages.
The ion source and subsequent ion optics can be operated to detect
positive or negative ions, and switching between these two modes within
an analytical run can be performed.
Under normal
conditions, ESI is considered a “soft” ionisation source, meaning that
relatively little energy is imparted to the analyte, and hence little
fragmentation occurs. This is in contrast to other MS ion sources, for
example the electron impact source commonly used in GC-MS, which causes
extensive fragmentation. It is possible to increase ESI “in-source”
fragmentation by increasing voltages within the source to increase
collisions with nitrogen molecules. This has been used in LC-MS analyses
to identify components with common structural features e.g. the glycans
in glycopeptides can be fragmented in-source to give 204 m/z
reporter ions. This feature has been used to identify glycopeptides in
tryptic digests of proteins in order to characterise the structure of
the glycans.
The following
popper user interface control may not be accessible. Tab to the next
button to revert the control to an accessible version.
Destroy user interface control6
Although useful for some analytes, in-source fragmentation is limited
for others, and more consistent fragmentation methods, such as collision
induced dissociation (see below), are required to induce extensive
fragmentation required for structural studies and tandem MS.
Small
molecules (≈ <500 Da) with a single functional group capable of
carrying electrical charge give predominantly singly charged ions. This
can involve the addition of a proton to the analyte (M+H+) when the ion source is operated in positive ion mode or the loss of a proton (M-H−) when operated in negative ion mode. Adduction of cations (e.g. M+NH4+, M+Na+, M+K+) and anions (e.g. M+formate−, M+acetate−)
can occur when salts are present. Larger molecules and molecules with
several charge-carrying functional groups such as proteins and peptides
can exhibit multiple charging, resulting in ions such as M+2H2+, M+3H3+
etc. For proteins, this results in an envelope of ions with different
charge states. This property can be used to accurately determine
analytes with high molecular weights including proteins up to 100 kDa on
mass spectrometers that scan up to only 4000 m/z. Indeed, it is unusual to detect ions with m/z values above this.
While
ESI is the most widely used ion source for biological molecules,
neutral and low polarity molecules such as lipids may not be efficiently
ionised by this method. Two alternative ionisation methods developed
for such analytes are described below.
Atmospheric Pressure Chemical Ionisation Source
In
atmospheric pressure chemical ionisation (APCI), as with ESI, liquid is
pumped through a capillary and nebulised at the tip. A corona discharge
takes place near the tip of the capillary, initially ionising gas and
solvent molecules present in the ion source. These ions then react with
the analyte and ionise it via charge transfer. The technique is useful
for small, thermally stable molecules that are not well ionised by ESI.
The
following popper user interface control may not be accessible. Tab to
the next button to revert the control to an accessible version.
No comments:
Post a Comment