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

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.