Critical Analysis of Mass Spectrometry

Introduction

1. Background:

The analytical technique that I have chosen to give an in-depth analysis of is Mass Spectrometry (MS)

This analytical technique is basically the study of ionised molecules in the gaseous phase; its main use is in the determination of the molecular weight of the molecule in the sample under investigation by accelerating ions in a vacuum environment. While this analytical technique has been around for over one hundred years there are significant advances being made to this technique in order to cater for more adverse samples which will be discussed in more detail later on. The main difference between mass spectrometry and other spectroscopy methods such as NMR is that it not dependant on transitions between energy states which may be responsible for its popularity. The diagram shown below (Figure 1.1.) [1] shows a simple diagram of a common mass spectrometer using electron ionization:

Figure 1.1 represents a schematic diagram of an electron ionization-mass spectrometer showing the various processes involved. Courtesy of www.molecularstation.com.

In its simplest form the process of determining the molecular weight of the sample typically occur over four main stages which are: Sample volatilisation, Ionisation, Separation and detection.

Sample volatilisation: The sample to be analysed if gaseous or volatile can be readily inserted into the mass spectrometer with the more solid samples requiring heating before insertation in order to construct a more volatile or gaseous sample. As can be seen form the above figure the sample is then moved further down the spectrometer towards the area where ionization of the molecules occurs.

Ionization: The sample is then hit with a barrage of high energy electrons from an electron gun with a charge of around 70 electron volts (eV). When the molecules collide with the high energy electron beam energy is transferred from the beam to the molecules which cause an acceleration of the molecules. These molecules may then dump an electron forming cation known as the molecular ion (M+•) [2]. This interchange is represented in the equation below (Figure 1.2.):

M+ e–M+• + 2e–

Moleccular Ion

This electron barrage usually results in most of the molecular ions fragmenting causing some of the fragments to not gain any charge and remain neutral and have no further part to play. The main purpose of ionisation is to donate a charge to the sample in order for the molecules to break up and become charged. The ionization method discussed here is electron ionisation however there are many other more methods of ionization which will be discussed in detail later on in my analysis.

Separation: This beam of newly charged molecular ions then proceed through a mass analyzer which in this case is a very strong controllable magnetic field which separates the charged molecules according to their mass to charge ratio (m/z) causing some of the molecules which are “too heavy” or “too light” to be thrown towards the top or bottom of the spectrometer and hence avoid detection. By varying the magnetic field, ions with different m/z values can be detected. Just like there are many different ionization methods for different applications there are also several types of mass analyzers which will also be discussed later.

A fundamental consideration in mass spectrometry at this point is mass resolution, defined as R = M/?M. where R is the resolution, M is the mass of particle and ?M is the mass difference compared to adjacent peak with overlap at 10% of peak height. Nowadays a magnetic sector analyzer can have R values of 2000-7000 depending on the instrument [3].

Detection: the final stage in the process is comprised of a detector which then amplifies and records the mass of the ions according to their m/z values. The detector may be set up for detection of molecular ions possessing different mass to charge ratios. The Molecular ions each have a mass that is almost identical to the mass of the molecule (M) and due to the fact that the charges on most of the molecules are usually 1, the value of m/z obtained for each of the ions is simply its mass. The data collected by the detector is fed to a recorder and is presented in the form of a plot of the numbers of ions versus their m/z values [3]. An example of this type of plot is shown below in figure 1.3. [4]:

Figure 1.3: A typical graph produced for a sample using mass spectrometry. Picture courtesy of www.research.uky.edu.

2. Methods of Ionization:

Electron Ionization (EI): as described above is the simplest method for converting the sample to ions and this method is found on the most common mass spectrometers. Many other simple and complex ionization methods exist for analyzing various samples. Some of these methods include:

Chemical Ionization (CI): This is a softer ionization method than EI, causing less fragmentation of the sample under investigation and hence it is mainly used for more sensitive compounds such as 2, 2-dimethylpropane for example which is prone to fragment with little stress. This decrease in fragmentation is due to the ions arising from a chemical reaction rather than bombardment and hence possesses less energy than those produced from EI. In Chemical ionization the molecules to be studied are mixed with an ionized carrier gas which is present in excess. Common carrier gases for CI include ammonia, methane, isobutene and methanol. The selection of the carrier gas depends on the degree of ion fragmentation required. Different carrier gases produce different mass spectra plots. The main advantage of CI is its softer approach lending to clearer results over EI for some samples. Other advantages include the relatively cheap and strong hardware as with EI. The main drawback of using chemical ionization in mass spectrometry is the fact that like electron ionization the sample must be readily vaporised in order for the molecules to gain that vital charge. This immediately dismisses the use of high molecular weight compounds and biomolecules [3]. It’s obvious therefore that CI and EI are very similar methods of ionization and due to this many of the modern day mass spectrometers can switch between these two methods effortlessly.

Electrospray Ionization (ESI): is a type of atmospheric pressure ionization. This technique is very useful for studying the high biomolecular weight molecules and other samples which may not be very volatile as discussed above. The sample to be investigated is sprayed through a fine capillary which has a charge on its surface, the sample then enters the ionization chamber resulting in the production of multiple charged ions along with single charged ions. This formation of multiple charged ions is very useful in the mass spectrometry analysis of proteins [3]. It is important to note that negative ions may also be formed in ESI and the operation may need to be reversed. ESI has become much more common over the last few years as it relies on a sample in solution which permits its use in LC-MS [5]. Thermospray Ionization (TSI) is closely related to ESI differing only in the fact that it relies on a heated capillary rather than a charged capillary; however ESI remains the more popular of the two methods.

Atmospheric-pressure chemical Ionization (APCI): It is obvious form the title that APCI is also a form of atmospheric pressure ionization resulting in a similar interface being used for both methods. This method was born in the 1970’s when it was first combined with liquid chromatography (LC) by Horning et al [6] who conveyed a new atmospheric ion source which used 63Ni beta emission in order to produce the required ions. Even tough APCI and ESI are harmonizing methods the main advantage APCI has over ESI is that it is more effective at determining the mass spectra for less polar compounds due to the reality that the gas phase ionization is more effective in APCI. Many MS instruments are now readily available with high mass resolution and accurate mass measurement, properties which are not as readily available with GC-MS instruments.

Fast Atom Bombardment (FAB): this type of ionization method is primarily used for large polar molecules. The sample to be studied is usually dissolved in a liquid matrix which is non-volatile and polar such as glycerol. This sample is then bombarded with a fast atom beam such as Xe– atoms which picks up electrons thus causing ionization from this reaction. This is a simple and fast method to use and is very good for high-resolution measurements. On the downside however it may be hard to compare low molecular weight compounds from the chemical back ground which is always high [5].

Desorption Chemical Ionization (DCI), Negative-ion chemical ionization (NCI), Field Ionization (FI) and Ion Evaporation are other less common ionization methods used in mass spectrometry.

3. Mass Analysers:

As described earlier the mass analyzers are used to separate the various ions according to their mass to charge ratio (m/z) and hence focus the ions with the desirable m/z value towards the detector. Some of the mass analyzers available include; Double-Focusing Mass Analyzers, Quadrupole Mass Analyzers, Time-of-Flight Mass Analyzers and Ion Trap Mass Analyzers.

Double-Focusing Mass Analyzers are used when a high resolution is of paramount importance. This high resolution is achieved by modifying the basic magnetic design. The beam of ions passes through an electrostatic analyser before or after the magnetic field causing the particles to travel at the same velocity resulting in the resolution of the mass analyzer increasing dramatically. Resolution may be varied by using narrower slits before the detector. It is important to note that this type of analyzer reduces sensitivity but increases accuracy resulting in a fine line between success and failure with regards to detection, for this reason this type of mass analyzer is only used for very selective purposes.

Quadrupole Mass Analyzers do not make use of magnetic forces for mass detection; instead they are composed of four solid rods arranged parallel to the direction of the ion beam. Using a combination of direct-current and radiofrequency the quadrupole separates the various ions according to their mass extremely quickly. Quadrupole mass analyzers are most on most GC-MS instruments.

Time-of-Flight Mass Analyzers (TOF) operate by measuring the time taken for an ion which has been produced to travel for the ion source to the detector [7]. This is based on the simple assumption that the lighter ions will have a greater velocity and thus will strike the detector first. This type of analyzer has become more and more common in recent years due to the fact that the electronics used in this analyzer have become much more affordable since it was first introduced in the 1940’s. In recent years the resolution and sensitivity of TOF have been increased by the insertation of a reflective plate within the flight tube itself [8]. The main area that this type of analyzer is used is in Matrix Assisted Laser Desorption Mass Spectrometry (MALDI-MS) discussed later.

The Ion Trap Mass Analyzer is composed of two hyperbolic end cap electrodes and a doughnut shaped ring electrode [7]. It is very similar to the quadrupole analyzer in resolution terms and basics however the ion trap is more sensitive.

4. The Mass Spectra:

The main interest that anybody has from the mass spectra is the molecular weight of the sample that was processed. The value of m/z at which the molecular ion (M+•) appears on the mass spectrum tell us the molecular weight of the original molecule. The most saturated ion formed from the ionization provides us with the tallest peak in the spectra know as the base peak (Figure 1.2). From this information the determination of very exact molecular weights of substances may be deduced which is probably the most important application of mass spectrometers. This determination also allows use to distinguish between different substances with a very similar molecular mass which we are unable to do ourselves. For example; the molecule C14H14 has a molecular mass of 182.1096 and the molecule C12H10N2 has a molecular mass of 182.0844. These two molecules may only be differentiated by MS as there is only 0.0252 in the difference even tough they are two completely different molecules. The type of MS instrument used in this case is a Double Focusing Mass Spectrometer as discussed briefly above which is capable of providing measurements accurate to 0.0001 atomic mass units. The chance of two compounds having the exact same mass spectra is very unlikely and therefore it is possible to identify an unknown compound by comparing its mass spectra obtained with that of a known library of mass spectra for various compounds.

5. Mass Spectrometry in Synergy with other Techniques:

Through the years mass spectrometers have evolved to be used not just on their own but used in tandem with a range of other analytic techniques such as Liquid Chromatography – Mass Spectrometry (LC-MS) in purity assessment and investigating rat urine, Gas Chromatography-Mass Spectrometry (GC-MS) for the detection and measurement of illicit drugs in biological fluids. It is LC-MS that has become the gold standard for detection and analyzation of substances. Gas chromatography works particularly well with mass spectrometry too, due to the face that the sample is already in its gaseous form at the interface. This system has been used by De Martinis and Barnes [9] in the detection of drugs in sweat using a quadrupole mass spectrometer which has been discussed earlier. The ability to identify metabolites in the biological fluids mentioned above can be very difficult and this is due to the fact that these metabolites are present in extremely low concentrations such as parts per million (ppm) or even less in some situations. For many years Nuclear Magnetic Resonance (NMR) was used to identify these metabolites but in recent times it would appear that mass spectrometry has become the more popular method for detection of the metabolite. This may be due to the fact that MS is more sensitive than NMR resulting in less sample amount being required.

6. Advances in Mass Spectrometry Instruments and their Limitations:

As mentioned briefly above it is very difficult to study large biomolecules such as proteins due to the fact that they are large polar molecules which are not volatile and as a result are difficult to convert to a gaseous state in order to undergo ionization. In recent years a solution to this problem has been accomplished with the introduction of Matrix- Assisted Laser Desorption Ionization (MALDI).

MALDI is a laser based soft ionization method which relies on the sample being dissolved in a solution containing an excess of matrix such as sinapinic acid which has a chromophore that absorbs at the laser wavelength, the sample is placed in the path of high intensity protons causing a collision of the atoms with the sample resulting in ionization of the sample molecules causing them to be ejected from the matrix. One of the main advantages of MALDI-MS is that only a very tiny amount of sample is required (1 X 10-5 moles) [3]. This technique has proven to be one of the most successful ionization methods for mass spectrometry analysis of large molecules due to its soft ionization ability. This technique has been used in the drug-biomolecule complexes in order to investigate the interaction properties and sites of biomolecules with various drugs on the market today [10]. This method was also used by Zschorning et al. to investigate the extracts of human lipoproteins after treatment with cholesterol esterase’s [11].

This method although very popular suffers some drawbacks. There is a strong dependence on the sample preparation method and any mistake made during sample preparation or any contamination introduced into the matrix during the sample preparation renders the rest of the investigation pointless. Another draw back of this method is the short sample life although some research has been undertaken [12] with the use of liquid matrices in the belief that this may increase the sample life by making use of the self-healing properties of the sample through molecular diffusion. One obvious drawback that may occur is the fact that the sample may not be soluble and hence may not dissolve in the matrix. This problem may be overcome with the use of compression of a finely ground sample and analyte [13]. Another disadvantage which may become of detrimental in the future is the fact that MALDI is not easily compatible with LC-MS, this problem may have to be rectified id the popularity of MALDI is to continue.

Electrospray Ionization (ESI) has been described in detail under the methods of ionization section above and it can be seen that this young technique is proving to be very useful with LC-MS to investigate the a variety of molecules including proteins, DNA and synthetic polymers.

The main problem with ESI-MS is that the mass spectra produced may contain many peaks of multiply charged ions which may cause confusion in the interpretation of spectra of some samples. The ESI instrument itself can also present with decreased sensitivity due to the presence of impurities such as salts and buffers, this is not the case with MALDI.

Although both MALDI and ESI are both very effective methods of developing mass spectra for large molecules such as proteins, MALDI still remains the method of choice for most analyses. However, as discussed above the fact that MALDI is not very compatible with LC-MS may pave the way for a surge in popularity of the LC-MS friendly ESI.

7. The Future of Mass Spectrometry:

Mass spectrometry has come along way since 1897 when Joseph J. Thompson used an early mass spectrometer to discover the electron and there is no reason why the mass spectrometer will continue to advance and evolve into the foreseeable future. The mass spectrometer is an extremely versatile analytical tool which can work in tandem and alongside other analytical methods such as chromatography seamlessly.

The main areas in which mass spectrometers have been used for quantification of compounds are LC-MS and GC-MS using the various ionization methods respectively. LC-MS is the gold standard in quantitative bioanalyses and is used by the majority pharmaceutical companies. The other minority tend to use other techniques such as High Pressure Liquid Chromatography (HPLC) and UV as they deem LC-MS to be too expensive.

An area of mass spectrometry to watch out for in the future is the use of ion-trap technology to perform LC-MS-MS to LC-MS [7]. This method already exists but reliable routine bioanalytical assays have not been produced as of yet.

References:

[1]http://www.molecularstation.com/molecular-biology-images/506-molecular-biology-pictures/21-mass-spectrometer.html

[2]Daniel C. Harris: Quantitative Chemical Analysis, sixth edition (2003) published by W. H. Freeman and Company, New York.

[3]Donald L. Pavia, Gary M. Lampman, George S. Kriz and James R. Vyvyan: Introduction to Spectroscopy, fourth edition, published by Brooks/Cole, Cengage Learing.

[4]www.research.uky.edu/ukmsf/whatis.html

[5]Ionization Methods in Organic Mass Spectrometry

[6]Horning, E.C., Caroll, D.J., Dzidic, I., Haegele, K.D., Horning, M.G., andStillwell,R.N. (1974). Atmospheric pressure ionization (API) mass spectrometry. Solvent-mediated ionization of samples introduced in solution and in a liquid chromatograph effluent stream, J. Chromatography. Sci, 12, (11), 725-729

[7]RF Venn (Ed) (2000) Principles and practice of Bioanalysis Taylor and Francis.

[8]Ashcroft, A.E. (1997) Ionisation Methods in Organic Mass Spectrometry, Cambridge, UK: The Royal Society of Chemistry.

[9]http://www.asms.org/whatisms/p1.html: The American Society of Mass Spectrometry

[10]Skelton, R., Dubols, F., Zenobl, R. Analytical Chemistry (2000), 72, 1707-1710

[11]Zschornig, Markus Pietsch, Roesmarie SuB., Jurgen Schiller and Michael Gutschow. Cholesterol esterase action on human high density lipoproteins and inhibition studies: detection by MALDI-TOF MS.

[12]Zenobi, R, Knochenmuss, R. Mass Spectrom, Rev. 1999, 17, 337-366.