A dissertation submitted to the Faculty of Science, University of the
Witwatersrand in fulfilment of the requirements for the degree of
Master of Science. University of the Witwatersrand, Johannesburg, March 2016 / A liquid phase microextraction based on single hollow fibre followed by liquid
chromatographic determination was developed for the extraction and
quantification of the hallucinogenic muscimol and its two precursors, tryptophan
and tryptamine from urine samples. A multivariate design of experiment was used
in which a half fractional factorial approach was applied to screen six potential
factors (donor phase pH, acceptor phase concentration, supported liquid
membrane composition, stirring rate, extraction time and salt content) for their
extent of vitality on the extraction of muscimol, tryptophan and tryptamine using
the developed method. Four factors were identified as essential for an enhanced
enrichment of each of the three research analytes from diluted urine samples.
The paired vital factors were then optimized using central composite designs
where empirical quadratic response models were used to visualize the response
surface through contour plots, surface plots and optimization plots of response
output. When the muscimol-based optimum factor levels were applied for the
simultaneous extraction of the three research analytes, a composite desirability of
0.687 was obtained implying that the set conditions were ideal for a combined
extraction of the analytes from the donor phase into the acceptor phase across a
supported liquid membrane impregnated with a carrier molecule. This was an
acceptable result considering that only the optimized muscimol factor levels were
set as universal factor values. Muscimol was the analyte of interest in this
research.
The composite desirability value was predicted by setting the extraction
conditions to 20% (w/w) di-(2-ethylhexyl) phosphoric acid (DEHPA) in dihexyl
ether (DHE) supported on the walls of a hollow fibre into a 200 mM HCl acceptor
phase inside the hollow fibre from a 20% (v/v) diluted urine donor phase spiked in
the 0.1 – 10 μg mL-1 analyte concentration range maintained at pH 4 and stirred at
800 rpm for 60 mins. Experimentally, average enrichments of 4.1, 19.7 and 24.1
were obtained for muscimol, tryptophan and tryptamine, respectively.
iv
The complexity of urine and the anionic nature of the carrier molecule embedded
on the supported liquid membrane resulted in interfering peaks that could not be
completely resolved from the analyte peaks. Thus matrix-based calibration curves
were used to address matrix effects.
Various statistical approaches were used to validate suitability of the developed
method for its potential use in quantifying muscimol and its precursors from urine
samples. These validation measures were used as a way of determining the
method’s ability to maintain the extraction process at equilibrium over a specific
range of analyte concentrations over a period of analyte existence in a urine
sample. The r² values of the matrix-based linear regression prediction models
ranged from 0.9933 to 0.9986. The linearity of the regression line of the matrixbased
calibration for each analyte was directly linked to the analyte enrichment
repeatability. Simultaneous analyte enrichment repeatability over a 0.1 – 10 μg
mL-1 analyte spiking concentration ranged from an RSD value of 8.3% to 13.1%.
Limits of detection were 0.021 μg mLˉ¹, 0.061 μg mL-1 and 0.005 μg mL-1 for
muscimol, tryptophan and tryptamine, respectively.
Other validation parameters that were considered included specificity (and
selectivity), accuracy, robustness, extraction range and system suitability. The
accuracy of the developed method was reported as the reproducibility of
enrichment factor values over six spiking concentrations used in constructing
matrix-based calibration curves. System suitability was limited to an HPLC-UV
approach. Method suitability was addressed through a comparative summary in
which the LOD, LOQ and r² values for the developed method were compared to
other methods that have been used to extract muscimol from urine samples. The
relevance or acceptability of the enrichment factor values obtained for the
extraction of the three analytes was achieved by comparison with enrichment
factor values of several compounds with similar polarity that have been extracted
from urine samples using carrier-mediated hollow fibre liquid phase
microextraction. / GR2016
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/21207 |
| Date | January 2016 |
| Creators | Ncube, Somandla |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | Online resource (xvii, 158 leaves), application/pdf |
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