A Quantitative MALDI-MSI Study of the Movement of Molecules in Biological Systems

RUSSO, Cristina (2019). A Quantitative MALDI-MSI Study of the Movement of Molecules in Biological Systems. Doctoral, Sheffield Hallam University.

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Abstract

The use of mass spectrometry imaging (MSI) for the analysis of 3D tissue models of human skin has been shown to provide an elegant label-free methodology for the study of both drug absorption and drug biotransformation. The main aim of the work presented in this thesis was to develop methodology for quantitative assessment of percutaneous absorption using matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). Quantitative assessment of the absorption of an antifungal agent, terbinafine hydrochloride, into the epidermal region of a commercial full thickness living skin equivalent model (Labskin) was used as a model system. Different approaches to generate robust and sensitive quantitative mass spectrometry imaging (QMSI) data were developed and compared. The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging provided a high-resolution method for the determination of terbinafine hydrochloride in Labskin. A quantitative assessment of the effect of adding a penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle was also performed, and data was compared to LC–MS/MS measurements of isolated epidermal tissue extracts. Comparison of means and standard deviations indicated no significant difference between the values obtained by the two methods. In this thesis the localisation of hydrocortisone hydrochloride in ex-vivo skin was also investigated. Hydrocortisone exhibits a low ionisation efficiency that makes its detection challenging with mass spectrometry techniques. An in-solution and on-tissue chemical derivatisation reaction using the Girard reagent T, a hydrazine based reagent, significantly increased the sensitivity and detection of the respective hydrocortisone-derivative using MALDI-MSI. In an additional study, MALDI-MSI was used to assess the metabolic activity in Labskin by employing the approach of "substrate-based mass spectrometry imaging" (SBMSI). Preliminary MALDI-MSI data detected the activity of the carboxylesterase 1 enzyme in the epidermal layer of skin. The MALDI-MSI data was supported by preliminary LC-MS/MS analysis. To investigate the reproducibility of the results future investigations are required.

Item Type:	Thesis (Doctoral)
Contributors:	Thesis advisor - Clench, Malcolm [0000-0002-0798-831X]
Additional Information:	Director of studies: Malcolm Clench
Research Institute, Centre or Group - Does NOT include content added after October 2018:	Sheffield Hallam Doctoral Theses
Identification Number:	https://doi.org/10.7190/shu-thesis-00268
Depositing User:	Colin Knott
Date Deposited:	02 Apr 2020 13:57
Last Modified:	03 May 2023 02:05
URI:	https://shura.shu.ac.uk/id/eprint/26097

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