Mass spectrometry (MS) is a technique used to measure the mass and give insights into the structure of a given molecule. Molecules must be in the gas phase for MS analysis and the must be ionised (either bofore or after entering the gas phase). Usually an electric field is applied to the gaseous ions to accelerate them and they are then separated by their mass to charge ratio (m/z).
There are many types of mass spectrometer arising from the different combinations of ionisation and detection methods. Electron impact is a method of ionisation for small volatile molecules that uses electrons to ‘knock off’ other electrons from the molecule of interest, creating a positively charged radical that may undergo further transformations in the gas phase. Chemical ionisation uses a reagent gas that is ionised and collided with the analyte transferring the charge. Matrix-‐assisted laser desorption (MALDI) uses laser light to vaporise the molecules of the matrix containing the analyte. The matrix is designed to strongly absorb the light of the laser, and it can then transfer its energy to the sample. A potential difference is then applied to the plume of analyte and the sample becomes ionised. This ionisation technique is much ‘softer’ than those previously described, and can be used for large molecules such as proteins.84,155
In the work carried out in this thesis, only electrospray ionisation (ESI) was used. ESI is a very soft ionisation technique that requires a liquid sample input and can be used for the analysis of small ions up to large proteins. Sample solution is flowed through a capillary to the tip where a potential difference is applied. The voltage applied partly determines the charge on the sample ions. The solution forms charged droplets from which, with the aid of the sheath gas (typically N2), the
solvent molecules are evaporated. As the droplets shrink, the desolvation becomes more rapid due to Coulombic forces that eventually overcome the cohesive forces and the analyte is released from the droplet solvent-‐free.155 Figure 1.24 shows an
illustration of the process.
Figure 1.24 An illustration of the electrospray ionisation process.156
As mentioned above different methods of ionisation can be coupled with different mass analysers, and either used on their own or in combination with each other. The following mass analysers were used to obtain data in this thesis.
One of the simplest mass analysers is a quadrupole. It consists of four parallel rods to which a constant voltage and an oscillating radio frequency are
applied to opposite pairs of rods. Ions pass through the rods and their direction of travel is controlled by the frequencies applied to the rods. Hence, certain ions may be excluded, depending on their m/z values.
Time-‐of-‐flight (TOF) analysers, as the name suggests, separate ions by the time taken for them to travel through a high vacuum drift region within the analyser called the drift region. Ions leave the ionisation chamber, and several thousands of times a second a voltage is applied adjacent to the sample stream, sending pulses of ions of the same kinetic energy into the drift region. The ions are then separated by their mass-‐to-‐charge ratios. To improve the resolution, more advanced instruments use a reflectron; this is a class of ion mirror that consists of electrodes with progressively higher charges applied to them. This, in effect, doubles the drift region and the resolving power of the analyser as well as focusing ions of the same m/z.
An ion-‐tap analyser shares some similarities with the quadrupole analyser. Radio frequencies and applied voltages are used to select the ions. They are then ‘trapped’ within the analyser and can accumulate before they are expelled towards the detector.
Tandem mass spectrometry (MS/MS) is another technique used in the course of this work. This term can be used to describe a number of ion fragmentation techniques, but in this case it refers to collision-‐induced dissociation (CID). For this process an ion of interest in selected and isolated, often using a quadrupole or an ion trap. An inert gas (in this case N2) is then introduced and
In terms of application to the study of metal complexes, high-‐resolution mass spectrometry has been used for the characterisation of complexes, to study their interaction with small biomolecules such as GSH,157 their interactions with
proteins,158,159 and their interactions with DNA.160–164
Aims of this work
In order to understand the mechanisms of action of metallodrugs, it is important to develop new methods for their characterisation, and in particular their speciation in solution. It is not only important to identify target sites, but also to determine the number and types of ligands which are bound to the metal at the target site, and hence to determine how the initial ligands in the metallodrugs are involved in activation and/or target reconition.
The specific aims were as follows.
1. To use HPLC to characterise the lipophilicity of a series of photoactivatable Pt(IV) anticancer complexes for which partition coefficients could not readily be determined by conventional octanol-‐water shake flask methods, and to attempt to correlate their lipophilicity with cancer cell uptake and biological activity.
2. To explore the use of chiral HPLC columns to separate enantiomers of organometallic anticancer complexes, especially Ru(II) and Os(II) arenes, and cyclopentadienyl Ir(III) complexes. Such separation is important for
biological testing and target site recognition, which can show a strong dependence of the chirality of drugs. In particular, the aim was to examine the stability of separated enantiomers and complexes with subtle difference in structure such as facial chirality.
3. To explore the use of new methods involving the coupling of photonic crystal fibres with high-‐resolution mass spectrometry that may be more efficient both in terms of the volume of analyte required (nanolitres), in terms of the extent of photochemical conversion, and the detection of short-‐lived photoproducts.