Mass Spectrometry

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DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

Our third and final analytical technique for discussion in this chapter does not fall under the definition of spectroscopy, as it does not involve the absorbance of light by a molecule.  In mass spectrometry (MS), we are interested in the mass - and therefore the molecular weight - of our compound of interest, and often the mass of fragments that are produced when the molecule is caused to break apart.

The basics of a mass spectrometry

There are many different types of MS instruments, but they all have the same three essential components.  First, there is an ionization source, where the molecule is given a positive electrical charge, either by removing an electron or by adding a proton. Depending on the ionization method used, the ionized molecule may or may not break apart into a population of smaller fragments. In the figure below, some of the sample molecules remain whole, while others fragment into smaller pieces.

Next in line there is a mass analyzer, where the cationic fragments are separated according to their mass. 

Finally, there is a detector, which detects and quantifies the separated ions.

One of the more common types of MS techniques used in the organic laboratory is electron ionization.  In the ionization source, the sample molecule is bombarded by a high-energy electron beam, which has the effect of knocking a valence electron off of the molecule to form a radical cation.  Because a great deal of energy is transferred by this bombardment process, the radical cation quickly begins to break up into smaller fragments, some of which are positively charged and some of which are neutral.  The neutral fragments are either adsorbed onto the walls of the chamber or are removed by a vacuum source.  In the mass analyzer component, the positively charged fragments and any remaining unfragmented molecular ions are accelerated down a tube by an electric field. 

This tube is curved, and the ions are deflected by a strong magnetic field.  Ions of different mass to charge (m/z) ratios are deflected to a different extent, resulting in a ‘sorting’ of ions by mass (virtually all ions have charges of z =  +1, so sorting by the mass to charge ratio is the same thing as sorting by mass).  A detector at the end of the curved flight tube records  and quantifies the sorted ions.

Looking at mass spectra

Below is typical output for an electron-ionization MS experiment (MS data in the section is derived from the Spectral Database for Organic Compounds, a free, web-based service provided by AIST in Japan.

The sample is acetone.  On the horizontal axis is the value for m/z (as we stated above, the charge z is almost always +1, so in practice this is the same as mass).  On the vertical axis is the relative abundance of each ion detected.  On this scale, the most abundant ion, called the base peak, is set to 100%, and all other peaks are recorded relative to this value. For acetone, the base peak is at m/z = 43 - we will discuss the formation of this fragment a bit later.  The molecular weight of acetone is 58, so we can identify the peak at m/z = 58 as that corresponding to the molecular ion peak, or parent peak.  Notice that there is a small peak at m/z = 59: this is referred to as the M+1 peak.  How can there be an ion that has a greater mass than the molecular ion?  Simple: a small fraction - about 1.1% - of all carbon atoms in nature are actually the 13C rather than the 12C isotope. The 13C isotope is, of course, heavier than 12C  by 1 mass unit.  In addition, about 0.015% of all hydrogen atoms are actually deuterium, the 2H isotope.   So the M+1 peak represents those few acetone molecules in the sample which contained either a 13C or 2H.   

Molecules with lots of oxygen atoms sometimes show a small M+2 peak (2 m/z units greater than the parent peak) in their mass spectra, due to the presence of a small amount of 18O (the most abundant isotope of oxygen is 16O). Because  there are two abundant isotopes of both chlorine (about 75% 35Cl and 25% 37Cl) and bromine (about 50% 79Br and 50% 81Br), chlorinated and brominated compounds have very large and recognizable  M+2 peaks. Fragments containing both isotopes of Br can be seen in the mass spectrum of ethyl bromide: 

Much of the utility in electron-ionization MS comes from the fact that the radical cations generated in the electron-bombardment process tend to fragment in predictable ways.  Detailed analysis of the typical fragmentation patterns of different functional groups is beyond the scope of this text, but it is worthwhile to see a few representative examples, even if we don’t attempt to understand the exact process by which the fragmentation occurs.  We saw, for example, that the base peak in the mass spectrum of acetone is m/z = 43.  This is the result of cleavage at the ‘alpha’ position - in other words, at the carbon-carbon bond adjacent to the carbonyl.  Alpha cleavage results in the formation of an acylium ion  (which accounts for the base peak at m/z = 43) and a methyl radical, which is neutral and therefore not detected.

After the parent peak and the base peak, the next largest peak, at a relative abundance of 23%, is at m/z = 15.  This, as you might expect, is the result of formation of a methyl cation, in addition to an acyl radical (which is neutral and not detected). 

A common fragmentation pattern for larger carbonyl compounds is called the McLafferty rearrangement:

The mass spectrum of 2-hexanone shows a 'McLafferty fragment' at m/z = 58, while the propene fragment is not observed because it is a neutral species (remember, only cationic fragments are observed in MS). The base peak in this spectrum is again an acylium ion.

When alcohols are subjected to electron ionization MS, the molecular ion is highly unstable and thus a parent peak is often not detected.  Often the base peak is from an ‘oxonium’ ion.

Other functional groups have predictable fragmentation patterns as well. By carefully analyzing the fragmentation information that a mass spectrum provides, a knowledgeable spectrometrist can often ‘put the puzzle together’ and make some very confident predictions about the structure of the starting sample.

 

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Gas Chromatography - Mass Spectrometry

Quite often, mass spectrometry is used in conjunction with a separation technique called gas chromatography (GC).  The combined GC-MS procedure is very useful when dealing with a sample that is a mixture of two or more different compounds, because the various compounds are separated from one another before being subjected individually to MS analysis.  We will not go into the details of gas chromatography here, although if you are taking an organic laboratory course you might well get a chance to try your hand at GC, and you will almost certainly be exposed to the conceptually analogous techniques of thin layer and column chromatography.  Suffice it to say that in GC, a very small amount of a liquid sample is vaporized, injected into a long, coiled metal column, and pushed though the column by helium gas.  Along the way, different compounds in the sample stick to the walls of the column to different extents, and thus travel at different speeds and emerge separately from the end of the column.  In GC-MS, each purified compound is sent directly from the end of GC column into the MS instrument, so in the end we get a separate mass spectrum for each of the compounds in the original mixed sample.  Because a compound's MS spectrum is a very reliable and reproducible  'fingerprint', we can instruct the instrument to search an MS database and identify each compound in the sample.

The extremely high sensitivity of modern GC-MS instrumentation makes it possible to detect and identify very small trace amounts of organic compounds.  GC-MS is being used increasingly by environmental chemists to detect the presence of harmful organic contaminants in food and water samples.   Airport security screeners  also use high-speed GC-MS instruments to look for residue from bomb-making chemicals on checked luggage. 

Mass spectrometry of proteins - applications in proteomics

Mass spectrometry has become in recent years an increasingly important tool in the field of proteomics.  Traditionally, protein biochemists tend to study the structure and function of individual proteins.  Proteomics researchers, in contrast, want to learn more about how large numbers of proteins in a living system interact with each other, and how they respond to changes in the state of the organism.  One very important subfield of proteomics is the search for protein biomarkers for human disease.  These can be proteins which are present in greater quantities in a sick person than in a healthy person, and their detection and identification can provide medical researchers with valuable information about possible causes or treatments.   Detection in a healthy person of a known biomarker for a disease such as diabetes or cancer could also provide doctors with an early warning that the patient may be especially susceptible, so that preventive measures could be taken to prevent or delay onset of the disease.

New developments in MS technology have made it easier to detect and identify proteins that are present in very small quantities in biological samples.  Mass spectrometrists who study proteins often use instrumentation that is somewhat different from the electron-ionization, magnetic deflection system described earlier.  When proteins are being analyzed, the object is often to ionize the proteins withoutcausing fragmentation, so 'softer' ionization methods are required.  In one such method, called electrospray ionization, the protein sample, in solution, is sprayed into a tube and the molecules are induced by an electric field to pick up extra protons from the solvent.  Another common 'soft ionization' method is 'matrix-assisted laser desorption ionization' (MALDI).  Here, the protein sample is adsorbed onto a solid matrix, and protonation  is achieved with a laser.

Typically, both electrospray ionization and MALDI are used in conjunction with a time-of-flight (TOF) mass analyzer component. 

The ionized proteins are accelerated by an electrode through a column, and separation is achieved because lighter ions travel at greater velocity than heavier ions with the same overall charge.  In this way, the many proteins in a complex biological sample (such as blood plasma, urine, etc.) can be separated and their individual masses determined very accurately.  Modern protein MS is extremely sensitive – very recently, scientists were even able to obtain a mass spectrum of Tyrannosaurus rex protein from fossilized bone! (Science 2007, 316, 277).

In one recent study,  MALDI-TOF mass spectrometry was used to compare fluid samples from lung transplant recipients who had suffered from tissue rejection to control samples from recipients who had not suffered rejection.  Three peptides (short proteins) were found to be present at elevated levels specifically in the tissue rejection samples.  It is hoped that these peptides might serve as biomarkers to identify patients who are at increased risk of rejecting their transplanted lungs.  (Proteomics 2005, 5, 1705).