Abstract
"This thesis explores further development of a liquid sampling technique called direct analyte probed-nanoextraction (DAPNe) to analyse animal tissues and cells. DAPNe uses a nanocapillary to sample analytes from a surface at micron dimensions, and has been previously used for the analysis of forensic traces and single cells.
DAPNe sampling is typically followed by mass spectrometry analysis. Sample introduction is conventionally via nanospray ionisation (NSI). This thesis describes research exploring the possibility of introducing liquid chromatography as an additional step into the workflow to improve precision and selectivity.
A comparison of the baseline sensitivity of DAPNe-NSI and DAPNe-LC-MS for drugs in homogenised tissue showed that the addition of LC-MS into the workflow can be used to improve precision compared with NSI, particularly if an internal standard is not available, or an untargeted analysis is to be carried out.
The DAPNe-LC-MS method was optimised for the detection of anti-tuberculosis drugs in tissues, by making improvements to the repeatability of the sampling area and transfer efficiency. The developed method was sufficient to probe the anti-TB drug bedaquiline in different compartments of a TB granuloma. Elemental imaging using particle induced X-ray Emission (PIXE) was used to validate the DAPNe measurements.
The DAPNe-LC-MS approach was further adapted and tested for the feasibility for providing spatially resolved lipidomics analysis. The technique was compared to laser capture microdissection (LCM), followed by LC-MS for providing spatially resolved lipid profiles. Whilst the LCM-LC-MS approach returned a greater number of lipid identifications, using DAPNe-LC-MS, certain diglycerides and triglycerides were more readily detected. Furthermore, it was found that DAPNe lipidomics can be used in sequence with elemental imaging, unlike LCM-LC-MS, which destroys the sample. This approach, DAPNe-PIXE, allowed the relationship between Fe and lipids to be probed in a TB granuloma.
Finally, the research described herein provided proof of concept for obtaining lipidomics profiles and measurement of anti-TB drugs in single cells."