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New capillary electrophoresis methods for the analysis of paralytic shellfish poisoning toxins

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Abdul Keyon, AS (2014) New capillary electrophoresis methods for the analysis of paralytic shellfish poisoning toxins. PhD thesis, University of Tasmania.

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Abstract

Paralytic shellfish poisoning (PSP) toxins, or usually termed as paralytic shellfish
toxins (PSTs), produced by marine and freshwater microalgae during algal blooms
can accumulate in filter-feeding bivalve shellfish. Early detection of PSTs in
shellfish is therefore important for food and public health safety. High performance
liquid chromatography (HPLC) methods with pre- or post-column oxidation for
fluorescence detection (FLD) and HPLC-mass spectrometry (MS) are the most
widely used instrumental analytical methods for PSTs, but are not easily miniaturised
for field-deployable portable analyser. Capillary electrophoresis (CE) can be
developed as an alternative method as it is compatible with miniaturisation, making it
an attractive method for a portable analyser for early on-site detection. In order to develop appropriate portable instrumentation for CE of PSTs, it is
necessary to develop appropriate methods. This was first done by developing CE
methods with different detection techniques namely ultraviolet (UV), capacitively
coupled contactless conductivity detector (C4D), MS, and FLD - making this the first
report of the use of C4D and an improved FLD detection for various PSTs with CE.
Due to the fact that most oxidised PSTs were neutral, micellar electrokinetic
chromatography (MEKC) was used in combination with FLD. The capillary zone
electrophoresis-UV (CZE-UV) and CZE-C4D methods provided better resolution,
selectivity and separation efficiency compared to CZE-MS and MEKC-FLD
methods. However, CZE-UV and CZE-MS methods did not provide sufficient
sensitivity to detect PSTs at the regulatory concentration limit, while CZE-C4D and
MEKC-FLD did show sensitivity below or close to the regulatory limit. The latter
most portable methods were evaluated for the screening of PSTs in a naturally contaminated mussel sample. MEKC-FLD was successfully used for PSTs screening
in the periodate-oxidised sample, whilst CZE-C4D method suffered from significant
interferences from sample matrix; a result that motivated further investigation of an
on-line preconcentration method to deal with the high conductivity sample matrix
and improve the sensitivity.
Therefore, CZE with C4D was examined with counter-flow transient
isotachophoresis (tITP). The tITP system exploited the naturally high sodium
concentration in mussel sample to act as a leading ion, in combination with one
electrolyte acting as terminating electrolyte (TE) and background electrolyte (BGE).
Optimisation of the BGE concentration, duration of counter-flow and injected sample
volume suitable for tITP resulted in sensitivity enhancement of at least two-fold over
CZE-C4D method developed in the first body of work. In particular, the modest gain
in sensitivity was achieved in the existence of a high concentration of sodium, a
sample matrix property that was problematic in previous method. This allowed the
analysis of PSTs in mussel sample at below or close to the regulatory concentration
limit. The pre-column periodate oxidation MEKC-FLD method described in the first body
of work enabled direct screening of PSTs in shellfish sample; however some toxins
produced multiple and/or identical oxidation products, affecting selectivity and
specificity of the method. The findings initiated investigation of CE with droplet
microfluidic post-column reaction system for the separation and FLD of PSTs. The
concept was that PSTs were separated using CZE and electrophoretically transferred
into droplets segmented by oil. Formation of droplets and electrical connection in the
CE-droplet microfluidic system were first evaluated. Depending on the total flow
rate of both aqueous and oil phases, nL-sized droplets could be formed having frequencies between 0.7-3.7 Hz. The use of an off-the-shelf micro cross for
positioning a salt bridge across the droplet flow from the separation capillary outlet
enabled the compartmentalisation of the analytes while maintaining the electrical
connection. Further, the potential of the system was investigated for post-column
oxidation of PSTs. Compartmentalised in the droplets, PSTs reacted with periodate
oxidant already present in the droplets, in which only a single peak for each PST was
detected by FLD.
Given that the general objective of this research study is to develop suitable CE
methods that can be implemented for on-site PSTs detection, the potentials of the
developed methods compatible with miniaturisation and portability have been
demonstrated. The CE methods with different detection techniques, combined with
an on-line preconcentration and ability to be coupled with post-column reaction
indicates the versatility of CE as alternative analytical method for PSTs.

Item Type: Thesis (PhD)
Keywords: Capillary electrophoresis, paralytic shellfish poisoning toxins, contactless conductivity detection, fluorescence detection, microfluific, shellfish
Copyright Information:

Copyright 2014 the Author

Date Deposited: 16 Mar 2015 00:05
Last Modified: 15 Sep 2017 01:06
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