The main focus of the PhD study was to develop new gel electrophoresis and ICP-MS based methods to analyze a wide variety of the bio-molecules such as proteins, phosphoproteins and metalloproteins etc. The tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) method is commonly used to resolve low molecular mass proteins, however, it requires a high percentage gel and a very complicated procedure to achieve this separation. This study describes a modification to tricine-SDS-PAGE to make it more effective for the separation of smaller proteins and for coupling to ICP-MS. The modified method employs low percentage PAGE gels and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. This modified method was applied to analyze phosphopeptides. Phosphopeptides are very small in size and difficult to separate using the other techniques such as Laemmli SDS-PAGE, original tricine-SDS-PAGE, immobilized metal affinity chromatography (IMAC), size exclusion chromatography (SEC) etc. In this study a simplified procedure is described based on modifying the original tricine-SDS-PAGE method. A comparative study showed that this modified method successfully resolved a digest mixture of very low to high molecular mass phosphopeptides/peptides. In off-line coupling of this method with ICP-MS, much better recoveries of the peptides from the gel were obtained as compared to traditional methods which indicate the compatibility of this modified method for quantitative studies. An on-line coupling of the modified system with ICP-MS was also demonstrated and it was applied for the separation, detection and quantification of phosphopeptides. Another application of this modified system was the separation of serum proteins. Blood serum contains five major protein groups i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. The separation of these five major proteins in a single gel is difficult to achieve using traditional methods. The modified system was shown to be superior for the separation of these serum proteins in a 7% (m/v) native-PAGE gel and a cellulose acetate membrane. A further study was carried out into controlling the factors that cause metal loss and protein fragmentation in SDS-PAGE. Using a reducing sample buffer, and heating to high temperatures (90-100ºC) in alkaline or acidic conditions may cause protein fragmentation and decrease the metal binding affinity. 70ºC was found suitable to prepare the sample at neutral, alkaline or acidic pH as no fragmentation observed. To prevent metal loss, the binding constant (log K) values of metal-amino acids, play the major role. Those metals which have high binding affinities with the amino acids in proteins can also be affected by the variation of the pH so prior information about pH to maintain the binding constant values is essential to minimize metal loss. This was observed in the loss of zinc, and to a lesser extent copper from human serum albumin (HSA) as measured by inductively coupled plasma mass spectrometry (ICP-MS). The method described above was applied for the separation and quantification of the serum proteins obtained from age-related macular degeneration (AMD) patients (where the AMD patients were from Moorfields Eye Hospital, London). Zn and Cu were quantified employing external calibration. Zn concentration showed variation whilst Cu did not show any significant variations in samples from AMD patients. A brief study of the interaction of cisplatin and oxaliplatin with HSA and transferrin was also performed. Cisplatin bound much faster than oxaliplatin with HSA. After 24 hours incubation, cisplatin showed a decrease in signal intensity which indicates that cisplatin binding decreases with time. Cisplatin binding with transferrin as compared to HSA was not significant, which could be the result of unstable Pt-transferrin complex formation. Oxaliplatin did not show high binding to either protein, perhaps due to the presence of the bulky, non polar DACH ligand.

The work presented in this thesis concerns the development and evaluation of new methods of sampling and analysis of organic pollutants in the indoor and outdoor environment. In Paper I, the development of a new method was reported for the determination of the brominated flame retardant tetrabromobisphenol A (TBBPA) in air using sampling with glass fiber filter and polyurethane foam (PUF), ultrasonic solvent extraction and liquid chromatography coupled to electrospray ionisation mass spectrometry (LC-ESI/MS). The MS fragmentation mechanism of TBBPA was thoroughly investigated and different acquisition modes were evaluated to achieve the most sensitive and selective detection. In Papers II and III, the potential use of Empore SPE membranes was evaluated for air sampling of volatile, semi-volatile and particle-associated organic compounds. Breakthrough studies conducted for 24h at air flows of 10- 20 L/min showed that the SPE membranes efficiently retains volatile and semi-volatile organophosphate esters and particles >10 nm. Effort was invested in the development of fast and environmental friendly methods, with low cost, for sample clean up and analysis. In Paper II, the sample preparation technique was dynamic solvent extraction with methanol coupled to LC-ESI/MS. The total run time per sample, including both extraction and separation, was less than 34 min, consuming only 1.6mL methanol. In Paper III, efficiency of selective extraction of polycyclic aromatic hydrocarbons from particulate matter sampled with Empore SPE membranes, using dynamic subcritical water extraction (DSWE) was investigated. Acceptable recoveries of the investigated compounds from reference material (SRM 1649a) were achieved. In Paper IV, the application of dynamic solid phase micro-extraction (SPME) air sampling was evaluated using, gas chromatography/positive ion chemical ionisation (GC/PICI) and tandem-MS detection for the determination of organophosphate esters in work environment.