For over a decade, diquaternary ammonium gemini surfactants have shown promise as non-viral gene delivery agents in both in vitro and in vivo systems. Their continued development, however, requires an understanding of their biological fate. The absence of identification and quantification methods that can achieve that goal is what drove the development of simple and rapid mass spectrometry (MS)-based methods; the focus of my Ph.D. dissertation.
Prior to the development of these MS-based methods, an understanding of the gas phase behavior of diquaternary ammonium gemini surfactants is required. The development of a universal fragmentation pathway for gemini surfactants was achieved using low resolution and high resolution MS instruments. Single stage (MS), tandem stage (MS/MS and quasi-multi-stage (quasi MS3) mass spectrometry analysis allowed for the confirmation of the molecular composition and structure of each gemini surfactant through the identification of common and unique mass to charge values. Understanding the fragmentation behavior allowed for the specific identification and/or quantification of gemini surfactants by MS-based methods; including liquid chromatography low resolution tandem mass spectrometry (LC-LR-MS/MS), fast chromatography low resolution tandem mass spectrometry, fast chromatography high resolution mass spectrometry, desorption electrospray ionization low resolution mass spectrometry and matrix assisted laser desorption ionization high resolution mass spectrometry.
We hypothesized that a LC-LR-MS/MS method would be the most effective quantitative method for the quantification of N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3) within PAM212 cellular lysate; achieving the lowest lower limit of quantification (LLOQ). Although the LC-LR-MS/MS method achieved a LLOQ suitable for analysis of G16-3 within PAM212 cell lysate, its limitations made it an inefficient method. In comparison, the four alternative mass spectrometry methods were faster, more efficient and less expensive than a conventional LC-LR-MS/MS method for the post transfection quantification of G16-3 within PAM212 cell lysate to be determined; 1.45 ± 0.06 μM. Future application of the universal fragmentation pathway and each MS-based quantification method will be beneficial for the future development of diquaternary ammonium gemini surfactants to further understand their post transfection fate.
Identifer | oai:union.ndltd.org:USASK/oai:ecommons.usask.ca:10388/ETD-2013-11-1289 |
Date | 2013 November 1900 |
Contributors | El-Aneed, Anas |
Source Sets | University of Saskatchewan Library |
Language | English |
Detected Language | English |
Type | text, thesis |
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