Defense Date

4-26-2005

Graduation Date

Spring 2005

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Mitchell E. Johnson

Committee Member

David W. Seybert

Committee Member

Jeffry D. Madura

Committee Member

David J. Merkler

Keywords

GC/MS, HPTLC, Primary fatty acid amides, SPE

Abstract

Primary fatty acid amides (PFAM's) are a novel class of bio-active lipids present in mammals in trace level. Selective isolation of PFAM's from lipid extracts is crucial for obtaining them in pure and concentrated form for interference-free instrumental detection and analysis. Synthesis of these commercially unavailable lipids is also important for method development and quantitative analysis by instrumental means. In this study a wide variety of long chain saturated and unsaturated primary fatty acid amides were synthesized and characterized. This list includes the positional and geometrical isomers of naturally abundant PFAM's. Two isotopically enriched PFAM's were also synthesized which are important for establishing the fragmentation patterns of the amides by mass-spectral analysis as well as for use as internal standards in quantitative analyses. The separation of saturated and some unsaturated amides was obtained using a non polar HP-5MS column. A more polar column, BPX70, was employed for the separation of the geometric and positional isomers of the unsaturated amides.

Two methods for the isolation of PFAM's from lipid extracts were developed and validated. First method is a high performance thin layer chromatography (HPTLC), which was found to be efficient in quick profiling of different lipid classes present in a total lipid extract. Any lipid class of choice can also be further analyzed by scraping it off the HPTLC plates. The second method is a solid-phase extraction (SPE) for selectively extracting the amides in a single fraction by a specific elution solvent from a normal phase column. This fraction can be dried and brought to the desired concentration for further instrumental analysis. Lowest mass of amides to be loaded and recovered by SPE was also optimized. Both HPTLC and SPE amide isolation methods were validated using the total lipids extracted from N18TG2 mouse neuroblastoma cell line. The detection limits of the amides by gas chromatography/mass spectrometry (GC/MS) were found to be in the order of 5-30 pg. PFAM's isolated from rabbit brain and heart tissues by these methods were identified as palmitamide, stearamide and oleamide. Trace amounts of linoleamide and eicosenoamide were also observed in brain tissues.

Format

PDF

Language

English

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