No description available.

The electrochemical detector provides a promising detection mode for capillary electrophoresis (CE) due to its excellent sensitivity, good portability, high selectivity, easy miniaturization, low capital and running cost. To widen its scope for determining trace analytes in complex samples, three dual-electrode detectors were fabricated to enable the determination of electro-inactive analytes, to assess co-eluted peaks and to give a large enhancement of the detection sensitivity by modifying electrode surface using multi-walled carbon nanotubes (MWNTs). To determine trace non-electroactive amino acids present in human tears, a serial dual-electrode detector was developed using an upstream on-capillary Pt film electrode to oxidize bromide to bromine at +1.0 V and a downstream Pt disk electrode to detect the residual bromine at +0.2 V after their reaction with amino acids eluted out from the separation capillary. The bromide reagent was introduced after CE separation by a newly designed coaxial post-column reactor fabricated onto the PMMA chip. Using optimized CE buffer containing 20 mM borate, 20 mM SDS at pH 9.8, L-glutamine, L-alanine and taurine were baseline separated with detection limits ranging from 0.56-0.65 μM and a working range of 2-200 μM for L-glutamine and of 2-300 μM for both L-alanine and taurine. Method reliability was established by close to 100% recoveries for spiked amino acids and good agreement between the measured and the literature reported amino acid concentrations in tears. For the determination of polyphenols in wine, a microchip-CE device was fabricated with a dual-opposite carbon fiber microelectrode operated in a parallel mode to assess peak purity. Under optimized conditions, (+)-catechin, trans-resveratrol, quercetin, (-)-epicatechin and gallic acid were baseline separated within 16 min with detection limits ranging from 0.031- 0.21 mg/L and repeatability of 2.0-3.3 % (n=5). The use of an opposite dual-electrode enables the simultaneous determination of peaks and measurement of their current ratios at +0.8 V and +1.0 V vs Ag/AgCl. The capability of using current ratio to identify the presence of co-migrating impurities was demonstrated in a mixed standard solution with overlapping (+)-catechin and (-)-epicatechin peaks and in a commercial red wine with interfering impurities. Matching of both the migration time and the current ratio reduce false positive and validate polyphenol quantitation in red wine. Lastly, a dual-opposite MWNTs modified carbon fiber microelectrode (CFME) was developed to determine the biomarkers (4-nitrophenol, 4-nitrophenyl-glucuronide and 4-nitrophenyl-sulfate) needed to assess exposure to methyl parathion. Use of the MWNTs modified CFME showed a much higher sensitivity than bare CFME, with a detection limit of 0.46 μM for 4-nitrophenol. Baseline separation of all three biomarkers was obtained within 31 min by a 45 cm long capillary under 12 kV in a 20 mM phosphate buffer at pH 7.0. The method developed was successfully utilized to determine low levels of biomarkers in human urine without using complex pretreatment steps and delivered recoveries ranging from 95.3 - 97.3% and RSDs within 5.8% (n=3). Using a parallel dual-electrode detector was shown to deliver reliable results with matching current ratios and comparable migration time to those obtained from biomarker standards. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This study focused on the development of an analytical method whereby creatinine, creatine and caffeine could be determined quantitatively. Urine is the preferred body fluid for the analysis of metabolites that the body excretes after administration of medicinal and illicit drugs. The detection of these metabolites depends on the volume of water the patient has drunk or, in criminal cases, the amount of water the suspect may deliberately add to their urine to dilute it. Creatinine, whose concentration in urine has been found to correlate with muscle mass, is chosen as an endogenous control substance against which the metabolite concentration is compared. While high performance liquid chromatography with ultraviolet detection (HPLC–UV) is commonly selected for the analysis, the quality of chromatography is affected by the fact that creatinine, being highly polar, is not retained in the reversed-phase columns. Furthermore, urine contains many polar substances that elute with the solvent front along with creatinine, thereby grossly affecting HPLC measurements. Hydrophilic interaction chromatography (HILIC) is a good alternative, although these methods generally require extensive sample preparation. Direct infusion electrospray ionization mass spectrometry (DI–ESI–MS) is ideally suited to highly polar compounds and was selected for this work. Pneumatically assisted ESI is preferred above the standard ionization method of atmospheric pressure chemical ionization (APCI) since pneumatically assisted ESI disperses the solution into ion-containing aerosol droplets which do not promote online conversion of creatinine to creatine. The objective of this study was to develop a simple and sensitive DI–ESI–MS method for the determination of various compounds in urine with creatinine as analytical reference compound and internal standard (IS). The analytical method development includes addition of 1-methyl-3-phenylpropylamine as a primary IS to standard solutions as well as to urine samples, followed by direct infusion of the sample into a mass spectrometer to determine the absolute concentrations of creatinine, creatine and caffeine. After appropriate instrument conditions were established, linear graphs of analyte-IS signal intensity ratios were obtained. The ratio of the concentration of the analyte (drug or metabolite) to that of creatinine (as IS) may be used to determine analyte concentration in artificial samples and/or urine. This method is not affected by change in fluid volume or adulteration of urine samples because the analyte-to-creatinine ratio remains unchanged. As part of this study, the developed DI–ESI–MS method was compared with an LC–UV–MS method developed for this purpose. / AFRIKAANSE OPSOMMING: Hierdie studie fokus op die ontwikkeling van ‘n analitiese metode waardeur kreatinien, kreatien en kaffeïen kwantitatief bepaal kan word. Uriene is die voorkeur liggaamsvloeistof vir die analise van metaboliete wat deur die liggaam, na administrasie van mediese en onwettige middels, uitgeskei word. Die deteksie van hierdie metaboliete hang van die volume water af wat die pasiënt gedrink het, of in strafbare gevalle, die hoeveelheid water wat verdagtes met opset by hul uriene gevoeg het ten einde dit te verdun. Daar is bevind dat die konsentrasie van kreatinien in uriene met spiermassa korreleer, derhalwe is kreatinien as ‘n interne kontrolemiddel gekies waarmee die metaboliet-konsentrasie vergelyk kan word. Hoë-druk vloeistofchromatografie met ultravioletdeteksie (HPLC–UV) word algemeen vir die analise van kreatinien ingespan, maar die gehalte van die chromatografie word deur die hoogs polêre aard van kreatinien beïnvloed en het swak retensie in omgekeerde-fasekolomme tot gevolg. Bowendien, uriene bevat groot hoeveelhede polêre middels wat saam met kreatinien in die oplosmiddelfront elueer en sodoende HPLC-bepalings uitermatig beïnvloed. Hidrofiliese interaksiechromatografie (HILIC) is ‘n goeie alternatief, ofskoon omvangryke monster-voorbereidings algemeen vereis word. Direkte inspuitelektrosproei-ionisasiemassaspektrometrie (DI–ESI–MS) is ideaal geskik vir hoogs polêre stowwe en is vir hierdie studie gekies. Pneumatiese hulp-ESI word bo die standaard ionisasie-metode van lugdruk chemiese ionisasie (APCI) verkies weens pneumatiese hulp-ESI se vermoë om die oplosmiddel in aërosoldruppels wat ione bevat, te versprei – sonder die aanlynomskakeling van kreatinien na kreatien. Die doel van hierdie studie was om ‘n eenvoudige en sensitiewe DI–ESI–MS-metode te ontwikkel wat verskeie stowwe in uriene kan bepaal deur kreatinien as analitiese verwysingsmiddel en interne standaard (IS) vir die opstelling van ‘n IS-kalibrasiekurwe te gebruik. Die analitiese metode-ontwikkeling sluit die gebruik van 1-metiel-3-fenielpropielamien as primêre IS in. Die IS word tot standaard oplossings en urienemonsters gevoeg, gevolg deur direkte inspuiting van die monster in ‘n massaspektrometer om die absolute konsentrasies van kreatinien, kreatien en kaffeïen te bepaal. Lineêre kurwes van die seinintensiteitsverhouding van analiet tot IS is verkry na gepaste instrumentkondisies vasgestel is. Die verhouding van konsentrasie van die analiet (middel of metaboliet) tot dié van kreatinien (as IS) mag gebruik word om die analietkonsentrasie in die standaard oplossings en/of urienemonster te bepaal. Die metode word nie deur veranderinge in die vloeistofvolume of verwatering van urienemonsters beïnvloed nie, weens die analiet-tot-kreatinienverhouding wat onveranderd bly. ‘n LC–UV–MS-metode is voorts ontwikkel om die ontwikkelde DI–ESI–MS-metode se data te vergelyk.