Capillary electrophoresis with laser-induced fluorescence detection used in metabolite profiling

Tseng, Hua-Ming (2009) Capillary electrophoresis with laser-induced fluorescence detection used in metabolite profiling. PhD thesis, University of Nottingham.

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Abstract

Novel and sensitive CE methods coupled with LIF and LINF detection using a series of separation modes have been developed for profiling organic metabolites containing amines and carbohydrates in mammalian and plant biofluids. In this study, metabolites containing an amine group were derivatized with 4-Fluoro-7-nitro- benzofurazan (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence detection (LIF). Under the optimized conditions most of the amine-containing metabolites in human biofluids such as plasma, urine and saliva could be identified by reference to standard compounds and the concentrations measured were found to be in agreement with literature values.

Furthermore, 17 carbohydrates including mono-, di- and oligosaccharides are also simultaneously derivatized via a two-step reaction involving reductive amination with ammonia followed by condensation with NBD-F. Under the optimized derivatization conditions all carbohydrates were successfully derivatized within 2.5 h and separated within 15 min using borate buffer (90 mmol L−1, pH 9.2). The method was applied to measure sugars in nanoliter volume samples of phloem sap obtained by stylectomy from wheat and to honeydew samples obtained from aphids feeding from wheat and willow.

Finally, an on-line sample concentration technique, sweeping-micellar electrokinetic chromatography (sweeping-MEKC), in conjunction with UV laser-induced native fluorescence detection (LINF) was developed and applied to the detection of native fluorescent analytes in biofluid samples such as plant phloem sap, human plasma and urine samples. The concentration limits of detection of analytes were in the range 7–100 nmol L−1, which were 250–3600-fold improvement for dopamine, DOPA and epinephrine compared with conventional capillary zone electrophoresis (CZE). The results indicated that a long-term limitation of relatively low detection sensitivity in CE-UV analysis arisen from the small injection volume and short optical path-length could be much improved, while no apparent loss in separation efficiency occurred.

Item Type:Thesis (PhD)
Supervisors:Barrett, D.A.
Faculties/Schools:UK Campuses > Faculty of Science > School of Pharmacy
ID Code:873
Deposited By:Mr. Hua-Ming Tseng
Deposited On:18 Feb 2010 13:54
Last Modified:18 Feb 2010 13:54

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