The major use of mass spectrometry is for RMM and structure determination. It is always utilized in conjunction with information from the other sources. The other applications comprise use in conjunction with the gas chromatography (GC) and HPLC to give structural information regarding the component of mixtures as they elute from the column. They are as well employed to a lesser extent as detectors in gas chromatography.
Gas Chromatography-Mass Spectrometry (GC-MS) :
Gas chromatography (GC) was the most primitive chromatographic method to be interfaced with the mass spectrometer. The original kind of gas chromatography had a packed GC column having a gas flow rate passing via it at ca. 20ml/min and the main problem was how to interface the GC without losing the mass spectrometer vacuum. This was resolved via the use of a jet separator, where the column effluent was passed across a very narrow gap between two jets and the highly diffusible carrier gas was largely removed, while the heavier analyte molecules crossed the gap devoid of being vented. The problem of eliminating the carrier gas no longer exists as GC capillary columns give a flow rate of 0.5-2 ml/min that can be directly introduced into the mass spectrometer without losing vacuum.
Ionization methods used in GC-MS:
There are three main kinds of ionization methods used in GC-MS.
a) Electron impact:
b) Positive ion chemical ionization (PICI): In this case the positively charged ions can either relate with the analyte or transfer a proton to the analyte.
c) The most general form of ionization occurring in the case of negative ion spectra is electron capture ionization. A reagent gas is employed to collide with it in such a way that their energies are reduced to <10eV. Molecules having a high affinity for electrons are capable to capture these low-energy thermal electrons. This is often loosely termed as NICI however as it doesn't involve formation of a chemical adduct, it is strictly chemical ionization. The two generally observed kinds of electron capture are illustrated below:
Fig: Electron Capture in NICI
Application of GC-MS in Impurity Profiling:
GC-MS has found a role in impurity identification in the pharmaceutical industry. Such impurities can occur either from the manufacturing procedure or from degradation of the drug.
Liquid Chromatography-Mass Spectrometry (LC-MS):
The interfacing of a liquid chromatography to a mass spectrometer proved much more complex than interfacing a gas chromatography as each mole of solvent introduced to the instrument generates 22.4l of solvent vapor even at atmospheric pressure. The method has made vast advances in the last 10 years and there are many kinds of interface available, the most successful of which are the Electrospray and atmospheric pressure ionization sources. A few interfaces employed in LC-MS are listed here.
The eluent from the column is vaporized and a part of the vapor (ca 1%) is transferred to the mass spectrometer and the rest of the vapor is pumped to waste. The spectra generated are similar to CI spectra as the presence of the solvent vapor having the sample decreases the energy of the ionization method and adducts can be formed by the solvent sensitive to the 10-9 g level; mass range up to 2000 amu.
b) Electrospray (ES) Ionization:
This is the most general LC-MS interface. Flow rates up to 1 ml/min however best at 200 µl/min or below. A charged aerosol is produced at atmospheric pressure and the solvent is principally stripped away by a flow of N2 gas. The charged molecules are drawn to the MS through electrostatically charged plates. This can be employed to find out both small molecules and molecules up to 200000 amu. Spectra can be simple, containing molecular ion only, or fragmentation can be induced through varying the cone voltage. ES ionization is more appropriate for polar molecules. The benefit of ESI is that large molecules which are not volatile adequate to evaporate by heating can be introduced to the gas phase. The aminoglycoside antibiotic kanamycin is an illustration of a very polar compound for which ESI is the ideal ionization method.
c) Atmospheric Pressure Ionization:
This process is very much similar to ES, however can operate at normal LC flow rates of 0.2-2ml/min. ES instruments can be simply transformed to run this method. Ionization is more analogous to CI, with the corona discharge producing ions like H3O+ and N2+ that promote the ionization of the sample. This process is complementary to ES as this interface will ionize less polar molecules.
d) Matrix-Assisted Laser Desorption with Time of Flight (MALDI-TOF):
This can be employed for very big protein > 200000MU. The sample is dissolved in a light-absorbing matrix; soft ionization is promoted via a pulsed laser; and ions are ejected from the matrix and accelerated by utilizing an electrostatic field to a field-free region. The lighter ions travel fastest. In order to enhance resolution, a device termed a 'reflectron' is employed to focus the kinetic energies of a population of a specific ion prior to its entering a field-free region. The length of time taken for ions to reach the detector gives their molecular weight (MW). The pulsed nature of the ionization makes sure that there is no overlap between spectra. MALDI-TOF is an ideal method for the characterization of the MW of large proteins.
The ion trap separates ion via capturing them in a circular electrode, where they orbit till they are ejected through a variation in voltage. The technology is developing fast and has benefits over a quadrupole in that ions can be trapped as tandem MS-type fragmentation is produced. This can filter out background whereas the ion of interest is retained in the trap before being further fragmented and ejected.
f) Tandem Mass Spectrometry:
As a soft ionization method like ESI generates very little diagnostic fragmentation, it is often employed in conjunction with tandem mass spectrometry. The kind of mass spectra obtained by employing collision induced dissociation (CID) in a tandem mass spectrometer are identical to those that are obtained under EI conditions. Generally, the molecular ion of the molecule is chosen (the precursor ion) via the first quadrupole. The chosen ion is then fragmented by employing a second quadrupole, to which argon gas is introduced that acts as a collision cell. The fragments generated (that is, product ions) are separated by using a third quadrupole. The method can at times be used devoid of chromatographic separation, making it a very fast method in areas like clinical screening for diagnostic marker compounds.
Use of LC-MS in Drug Metabolic Studies:
The body metabolizes foreign compounds (that is, xenobiotics) like drugs to make them more polar and water soluble to facilitate the excretion from the body. LC-MS can be employed to recognize these metabolites.
Drug discovery comprises a number of phases, comprising target identification, lead identification, small molecule optimization and pre-clinical and clinical development.
Target recognition has been speeded up as an outcome of genomics however the measurement of gene transcription via detection of RNAs doesn't essentially point out precisely what the structures of the proteins produced are, as the proteins might be modified after translation through processes like glycosylation or phosphorylation. Advances in the mass spectrometry have allowed recognition of translated proteins. Such proteins might signal disease processes, in which case their regulation through a potential drug might point out its efficacy, not equally expression of some proteins following drug therapy might point out drug toxicity.
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