Forensic toxicology is a critical field in which scientific techniques are employed in order to establish the presence or absence of pharmacological substances and/or their metabolites within an individual. The results of such analyses can have legal implications, and toxicology has a number of important applications, including post-mortem investigations, workplace drug testing, therapeutic drug monitoring, and impaired driving studies. The focus of this specific body of work is on the use of toxicology in the detection and quantification of drugs of abuse –specifically opioids - in biological samples. In recent years, there has been a surge in opioid abuse and the need for forensic toxicology labs to process samples from such cases quickly and accurately continues to increase. As a result, it is imperative to research different techniques and technologies that can be applied in toxicology to improve efficiency of sample processing while still remaining sensitive and specific. Many toxicology laboratories today use immunoassay techniques for screening, and utilize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification. While these methods are established and reliable, the need to analyze an increasing number of samples in a more efficient time frame is essential, and with that, the need to develop and validate new analytical methods. This study sought to validate the use of Microchip Capillary Electrophoresis-Tandem Mass Spectrometry (CE-MS/MS) as a method for detecting and quantifying a panel of fourteen opioids. The experiments were run using a ZipChip (908 Devices, Boston, MA) as the separation scheme, which contains a small capillary where analytes are separated out by electrophoretic mobility - dictated largely by size and charge. These analytes were then ionized by electron spray ionization (ESI) at the end of the chip, and then detected, fragmented, and analyzed in a SCIEX 4500 Triple Quadrupole Mass Spectrometer (Framingham, MA). The analytical run time of the method evaluated was two and half minutes per sample. Calibration curves were run and the method was assessed for a number of validation parameters, including bias, precision, limit of detection, common analyte interferences, matrix interferences, and carryover, as recommended by the American Academy of Forensic Sciences Standards Board. The fourteen drugs and metabolites looked at in this study were 6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine (EDDP), fentanyl, heroin, methadone, morphine, naloxone, norfentanyl, oxycodone, oxymorphone, and tramadol. All standards were ordered from Cerilliant (Round Rock, TX), as well as deuterated internal standards used for quantification purposes. This study showed that as the method currently stands, it can reliably detect this panel of opioids at limits of detection between 1 and 15 ng/mL, with the exception of buprenorphine and morphine, for which the method appeared less sensitive. While some applications desire higher sensitivity than this, this level of detection could be very useful as a screening technique that is quick and also far more specific than current immunoassay screening techniques, and provide the additional advantage of quantification for samples at slightly higher concentrations. Quality control samples at 100 ng/mL and 150 ng/mL generally showed consistent results and acceptable levels of bias and precision, indicating that the method can be used to reliably quantify this panel of opioids at those concentrations. In addition, interference signals detected during analysis of other common analytes often encountered with opioids were negligible, with the exception of heroin and norfentanyl. Analysis of ten lots of urine for blank matrix interferences also demonstrated low potential for interference, with the exception of heroin. Finally, there was no evidence of significant carryover between samples, or interference from the deuterated internal standards. While some potential instrumentation issues such as mass spectrometer calibration prompt further study, the method shows promise for future use as a high throughput analysis tool in forensic toxicology labs. CE-MS/MS has the added benefit of not only faster run times, but significantly less sample consumption per run, and additionally, less sample preparation. CE is a viable separation scheme for metabolites and forensic applications, and could make large impacts as an effective way to analyze toxicological samples.

No description available.

This work is a contribution to the minimization of some of the selectivity and detection limitations in capillary electrophoresis. A more practical design of an electrochemical detector is introduced with simultaneous on-line UV detection¹, for the selective detection of a number of pungent and neurological compounds, the piperines and the capsacinoids. Commercially available microelectrodes together with large 25 μm id fused silica capillary columns are used for the first time in the presence of an auxiliary electrode. Minimum detectable quantities and efficiencies are sample dependent and were found to be comparable to the earlier more laborious electrochemical cell designs. To exploit the benefits of common additives that enhance the selectivity of electrolyte systems, various additives including α, β and γ Cyclodextrins, organic modifiers, as well as a series of cationic surfactants are explored for the separation of a number of industrially important isomeric aromatic carboXylic acids². The separation was found to depend largely on the analyte1s geometry, degree of ionization as well as on the buffer pH and composition. The resultant separations were compared for best efficiency, resolution and ruggedness. In addition, to add to the arsenal of CE selectors, a number of new micellar systems are investigated. Oligomeric sodium 10-undecylenate, a recently introduced oligomeric surfactant³ is structurally investigated through the separation of vitamins and the resultant selectivity and resolution is compared to the more commonly used surfactant sodium dodecyl sulfate⁴. Additionally, a number of phospholipids and Iysophospholipids, common constituents of cell membranes, are investigated not only as possible MECC surfactants but also as highly hydrophobic analytes needing themselves separation⁵. Finally, as a contribution to methods development, the effect of variations in systemparameter conditions is examined in a successful separation of a number of enzymes. / Ph. D.

In Parkinson's disease, severe damage to nigrostriatal neurons causes a depletion of the neurotransmitter dopamine (DA). Oxidative stress on the brain is thought to contribute to neuron cell death and to the onset of Parkinson's disease. Reactive oxygen radicals produced during oxidative stress have been implicated as an initiator of neuron destruction. Glutamate, an excitatory neurotransmitter, can initiate OH radical formation when present in excess. Oxidative stress on the brain caused by glutamate overflow may be monitored by trapping the OH radicals with salicylic acid to produce 2,3-dihydroxybenzoic acid (2,3-DHBA). Determination of this product is initially performed using capillary zone electrophoresis (CZE) coupled with UV detection to establish optimum separation conditions. These conditions were applied for rapid, efficient, and sensitive determination of 2,3-DHBA by CZE coupled with electrochemical detection. Quick and sensitive detection of 2,3-DHBA is essential in monitoring OH radical generation and identifying its role in Parkinson's disease. / Master of Science

The final goal of this study is to establish a microscale analysis method that allows solution phase characterization of interactions between β2-glycoprotein I (β2gpI) and some of its ligands. Human β2gpI is a phospholipid- and heparin-binding plasma glycoprotein. The physiological role of the protein in normal blood coagulation is not entirely known, nor is its role in autoimmune diseases characterized by blood clotting disturbances (thrombosis). Quantitative binding data of β2gpI interactions with some of its ligands may help elucidating the mechanisms behind these diseases and in the development of new approaches for diagnostics, prevention, and therapy. In this thesis, capillary electrophoresis (CE) was used as methodological platform for the interaction studies. The analysis of peptides and proteins by CE is desirable due to low sample consumption, possibilities for non-denaturing and highly effective separations. The first objective of this thesis was to find an approach to prevent charge dependent adsorption of β2gpI to the inner surface of the capillaries. Analyte adsorption at the negatively charged inner surface of fused silica capillaries is detrimental to interaction analyses. This phenomenon is especially pronounced in the analysis of basic proteins and proteins containing exposed positively charged domains, such as β2gpI. A new strategy to suppress these solute-wall interactions was devised, investigated and optimized. This strategy exploits the pH hysteresis behavior of fused silica surfaces, by simply performing an acidic pretreatment of the capillary. The results in this thesis show that the acidic pretreatment efficiently prevents protein adsorption. / <p>Papper 4 Estimation of the amount of β₂-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment ingick som manuskript i avhandlingen, nu publicerad.</p>

Au cours de cette thèse, nous avons été amenés à développer des méthodes analytiques afin de contrôler la qualité de protéines thérapeutiques en tant que produits finis ou d'évaluer le potentiel de certaines protéines à être des candidats médicaments. Deux protéines ont été étudiées : l'albumine de sérum humain (HSA) et l'antithrombine (AT). Ces protéines diffèrent par leur structure, leur glycosylation et leurs activités biologiques. Une méthode d'électrophorèse capillaire de zone (CZE) a permis de séparer neuf formes dans des préparations commerciales d'HSA plasmatique, dont des formes native, cystéinylée, glyquées et tronquées. Une autre méthode CZE a permis de séparer plus de huit formes dans des préparations commerciales d'AT plasmatique, dont des formes native, latente et hétérodimérique. Les deux méthodes CZE développées ont permis de mettre en évidence des différences significatives quant à la composition de préparations commerciales d'HSA ou d'AT produites par cinq fournisseurs différents. Des méthodes d'électrophorèse capillaire couplée à la spectrométrie de masse (CE-MS), avec un analyseur quadripôle-temps de vol (Q-TOF) et une ionisation électrospray (ESI), ont également été développées. Ces méthodes CE-MS ont permis d'identifier non seulement de nombreuses glycoformes et formes apparentées de l'AT, mais également des formes dimériques. Une méthode CE-MS en conditions non-dénaturantes a permis de montrer que la forme native et la forme latente de l'AT (deux conformères) présentaient un spectre de masse spécifique, permettant de les différencier sans ambiguïté. Afin d'évaluer l'affinité de variants d'AT pour l'héparine, une méthode d'électrophorèse capillaire d'affinité (ACE) a été développée. Cette méthode ACE a permis d'analyser les variants d'AT directement à partir des surnageants de culture cellulaire dans lesquels ils sont produits. Ainsi, il a été montré que certains variants présentaient une affinité très élevée pour l'héparine, en accord avec les mutations réalisées. Enfin, dans le cadre des études d'affinité entre l'AT et l'héparine, nous avons exploré une méthode nouvelle basée sur l'analyse de la dispersion de Taylor (TDA). / During this Ph.D., we developed analytical methods in order to check the quality of therapeutic proteins as finished products or to assess the potential of some proteins to be drug candidates. Two proteins were studied : human serum albumin (HSA) and antithrombin (AT). These proteins are very different regarding their structure, glycosylation, and biological functions. A capillary zone electrophoresis (CZE) method enabled to separate nine forms in commercial preparations of HSA issued from human plasma, among which native, cysteinylated, glycated, and truncated forms. Another CZE method allowed separating more than eight forms in commercial preparations of AT issued from human plasma, among which native, latent, and heterodimeric forms. Both CZE methods enabled to highlight significant differences in the composition of marketed preparations produced by five competitive pharmaceutical companies. Capillary electrophoresis-mass spectrometry (CE-MS) methods, using a quadrupole-time of flight (Q-TOF) analyzer and electrospray ionization (ESI), were also developed. These CE-MS methods enabled to identify not only a high number of glycoforms and related forms of AT, but also dimeric forms. A CE-MS method developed in non-denaturing conditions showed that native and latent forms of AT (two conformers) exhibited a specific mass spectrum, allowing to unambiguously distinguish them. With the aim to assess the binding affinity of AT variants toward heparin, we developed an affinity capillary electrophoresis (ACE) method. This ACE method enabled to analyze AT variants directly from the cell culture supernatants used to produce them. It has been shown that some AT variants had a very high affinity for heparin, in good agreement with the performed mutations. Finally, a new method based on Taylor Dispersion Analysis (TDA) was investigated to study the affinity between AT issued from plasma and heparin.

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Chemistry, Durban University of Technology, 2012. / The present work involves the interaction studies of chiral compounds with the Human Serum Albumin (HSA) protein using computational and experimental methods. The HSA protein has multiple binding sites that forms the basis for its exceptional ability to interact with many organic and inorganic molecules, which makes this protein an important regulator of intercellular fluxes and the pharmacokinetic behaviour of many drugs. This study was undertaken to evaluate the related pharmacokinetic and enantioselective binding parameters of the racemic catechin enantiomers with the HSA. Accordingly, this work involved a method development for the chiral separation of a racemic compound, by capillary electrophoresis-electrokinetic chromatography (CE-EKC) with a highly sulphated beta-cyclodextrin (HS--CD) as a chiral selector. The experimental work was supported by two molecular docking studies. The first included the mimicking of the host-guest interactions between a chiral selector and an enantiomeric compound. The second study included the estimation of the pseudo enantioselective (ES) binding of catechin to HSA. Overall, it was found that CE-EKC is the preferred method for the(±)-catechin binding to HSA protein evaluation. Moreover, the technique used in this work is not restricted to HSA or polyphenols, but can also be applied to other proteins and ligands that possess chirality. Furthermore, the molecular docking approaches also proved to be very useful for the evaluation of chiral recognition systems and for elucidation of the ligand-protein interactions.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Capillary electrophoresis (CE) has recently attracted considerable attention as a promising analytical technique for the separation of cations and anions in complex matrices. Determination of ions in aqueous samples using capillary electrophoresis can be accomplished with indirect UV detection. Most inorganic ions have weak absorption profiles in the UV-Vis wavelength range. These mostly non-absorbing species are commonly detected by indirect UV absorbance through addition of an absorbing co-ion (chromophore) into the electrolyte. Inorganic cations most often require an additional complexing agent to selectively alter their similar mobilities and proper separation. For optimal determination of alkali, alkaline, and transition metal ions, several electrolytes systems were studied. These include pyridine, imidazole and 4- aminopyridine as UV-absorbing species and glycolic acid, a-hydroxyisobutyric acid and their mixture were used as complexing reagents. A mixture of 10 metal ions (K+, Na+,Ca2+, Mg2+, Mn2+, Fe2+, Cd2+, Pb2+, Ni2+and Zn2+) was successfully separated. Detectionwas performed at 210,214 and 254 nm. In the anion determination chromate and 2, 6 pyridine dicarboxylic acids (PDC) were used as back ground electrolytes for inorganic ions (F-, CI- en SO₄² ̄ ) and organic acids (tartaric acid, malic acid, succinic acid and citric acid) separations respectively. Electroosmotic flow (EOF) was reversed in the direction of the anode by adding Cetyltrimethylammonium bromide (CTAB) to the electrolyte. Highly alkaline conditions were used to confer a negative charge on inorganic and organic anions to promote their migration towards the anode. The detection wavelength was 200 nm. All peaks were completely resolved and well separated. The limit of detection (LOD) of cations and anions were in the range of 0.5 - 3 ppm and 2 - 3.5 ppm respectively. The described methods were used successfully in routine analysis of real samples. This includes the qualitative and quantitative analysis of an environmental water samples from the areas surrounding Stellenbosch, beverages and orange juice. / AFRIKAANSE OPSOMMING: Kapillêre Elektroforese (CE) het in die onlangse verlede heelwat aandag getrek as "n belowende analitiese tegniek vir die skeiding van katione en anione in komplekse monsters. Die bepaling van ione in waterige medium met kapillêre elektroforese word gedoen deur indirekte Ultraviolet (UV) deteksie aangesien meeste anorganiese ione swak absorbsie in die die sigbare UV (UV-Vis) golflengtegebied toon. Deteksie van hierdie meestal nie-absorberende spesies word algemeen gedoen deur indirekte UV absorbansie deur die byvoeging van "n ko-ioon (chromofoor) tot die elektroliet. Anorganiese katione benodig dikwels "n addisionele komplekserings reagens om selektief hulle eenderse mobiliteite te verander en sodoende goeie skeiding te bewerkstellig. Vir die optimale bepaling van alkali-, alkali-aard- en oorgansmetaal ione is verskeie elektrolietsisteme bestudeer. Hierdie sluit in piridien, imidasool en 4-aminopiridien as UV absorberende spesies en glikoliensuur, a-hydroksie-isobottersuur asook "n mengsel van die twee as komplekserings reagense. "n Mengsel van 10 metaalione (K+, Na+, Ca2+, Mg2+, Mn2+, Fe2+, Cd2+, Pb2+, Ni2+ en Zn2+) is sukselvol op hierdie wyse geskei. Deteksie is gedoen by golflengtes van 210, 214 en 254 nm. Vir die anioon bepaling is chromaat en 2,6-piridiendikarboksielsuur gebruik as agtergrond elektroliete vir die skeiding van anorganiese anione (F-, CI- en SO₄² ̄ ) en organiese sure (tartaarsuur, malonsuur, suksiensuur en sitroensuur), onderskeidelik. Elektroosmotiese vloei (EOF) is omgekeer na die rigting van die anode deur byvoeging van setieltrimetielammoniumbromied (CTAB) by die elektroliet. Sterk alkaliese kondisies is gebruik om 'n negatiewe lading op die anorganiese en organiese anione te konsentreer en sodoende hul migrasie na die anode te bevorder. Die deteksiegolftengte hier gebruik was 200 nm. Volkome resolusie en goeie skeiding is gerealiseer vir al die pieke. Die deteksielimiete (LOD) vir die katione en die anione was 0.5 - 3 ppm en 2 - 3.5 ppm, onderskeidelik. Die metodes wat beskryf word is suksesvol aangewend vir roetiene analise van werklike monsters. Dit sluit in kwalitatiewe sowel as kwantitatiewe analise van omgewingswater monsters uit die Stellenbosch area, koeldranke en lemoensap.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: This study was initialized to introduce capillary electrophoresis (CE) as a useful technique in the analysis of fluid inclusions in quartz. lts advantages are low detection limits for the dissolved ionic content of the fluid, the small amount of sample (1 g or less) for a detailed qualitative and quantitative analysis, and the short time required to obtain results (one run for either cations or anions take approximately 10 minutes). The study area from which quartz veins were selected is situated within the Neoproterozoic Saldania belt. Syn- and post-tectonic S-, 1- and A-type granitoids from the Cape Granite Suite intruded the metamorphosed Malmesbury greywacke and pelites between 550 and 510 Ma. Additional periods of tectonism and metamorphism occurred during Cape Supergroup sedimentation (480 - 400 Ma) as well as Karoo sedimentation and the simultaneous Cape Orogeny (280 -215 Ma). The quartz-biotite±chlorite vems are hosted by Cape Granite as well as Malmesbury sediments. These barren quartz veins are part of two vein sets, one dipping at an angle between 15 and 500 to the S to SE and striking W, similar to Sn-mineralized quartz veins in the SW-cape, while the other is near-vertical and striking W to NW. Except for their orientation, no differences regarding associated minerals, inclusion characteristics or fluid chemistry indicated a difference in origin. Four fluid phases within a temperature range of 160 - 390 °C were identified as being largely late-magmatic and released from the underlying Cape Granite plutons, namely an early 370- 390 °C population, followed by the 310 - 360, 230 - 300 and lastly the 160 - 200 °C populations. Initiation of this fluid system occurred from at least SlOMa, after final granite intrusion, but the age of the final stage is unknown. Renewed fluid circulation occurred during a later period of metamorphism, possibly during the Cape Orogeny. These fluids had temperatures between 240 and 360°C and are of sedimentary origin, most likely released from the Malmesbury metamorphites. The technique of capillary electrophoresis has been evaluated for its application to bulk fluid inclusion analysis, and the crush-leach fluid extraction procedure of Bottrell, et al., (1988) optimized for CE analysis. Contamination factors were identified and minimized or eliminated, where possible. Bulk fluid inclusion chemistry obtained by CE was therefore proved to provide valuable information regarding the various fluid generations as long as inclusion populations are investigated individually to explain and correlate bulk data. / AFRIKAANSE OPSOMMING: Die doel van die studie was om te toon dat die tegniek van kapillêre elektroforese bruikbaar is in die analiese van vloeistofmsluitsels in kwarts. Die voordele van hierdie tegniek is lae deteksie limiete vir die opgeloste ioon inhoud van die vloeistof, die klein monstergrootte (< 1g) wat nodig is vir 'n omvattende kwalitatiewe en kwantitatiewe analise, en die kort tydsduur waarin resultate verkry word ('n katioon of anioon analise vir een monster duur lO minute). Die studie gebied waar kwarts are gemonster is, is binne die Neoproterosoïese Saldania Gordel geleë. Sin- en laat-tektoniese S-, I- en A-tipe graniete van die Kaapse Graniet Suite het die gemetamorfiseerde Malmesbury grouwakke en peliete tussen 550 en 510 Ma binnegedring. Latere periodes van tektonisme en metamorfose het tydens deponering van die Kaap Supergroep (480 - 400 Ma), en die gelyktydige episodes van Karoo sedimentasie en Kaapse Orogenese (280 - 215 Ma) plaasgevind. Die gasheer gesteentes vir die kwarts-biotiet±chloriet are is Kaapse Graniet sowel as Malmesbury sedimente. Hierdie ongemineraliseerde are is deel van twee aarstelsels, nl. een met 'n duik hoek tussen 15 en 50° S tot SO en 'n westelike strekking, soortgelyk aan die Sn- ,.gemineraliseerde are in die SW-Kaap, terwyl die ander stel are feitlik vertikaal is en W tot NW strek. Behalwe vir die verskil in oriëntasie was daar geen aanduiding, wat betref 'n verskil in geassosieerde minerale, vloeistofinsluitsel kenmerke of vloeistof chemie, dat hierdie twee aarstelsels van verskillende oorsprong is nie. Vier vloeistof fases binne 'n temperatuur gebied van 160 - 390 °C en 'n vloeistof saliniteit van 0 - 5.7 gewig % NaC1 ekw. is geïdentifiseer, met 'n laat-magmatiese assosiasie en vrygestel deur die onderliggende Kaapse Graniete. Dit behels 'n vroeë 370 - 390 °C populasie, gevolg deur die 310 - 360, 230 - 300 en laastens die 160 - 200 °C populasies. Inisiasie van hierdie sisteem kon moontlik rondom 510 Ma gelede plaasgevind het, maar die ouderdom van die finale fase is onbekend. Hernude vloeistof sirkulasie het tydens 'n later stadium van metamorfose onstaan, moontlik tydens die Kaapse Orogenese. Hierdie vloeistowwe het temperature tussen 240 en 360 °C en is van sedimentêre oorsprong waar dit moontlik deur metamorfose van die reeds gemetamorfiseerde Malmesbury gesteentes vrygestel is. Die tegniek van kapillêre elektroforese is vir die toepassing daarvan in die analise van vloeistof insluitsels in kwarts geëvalueer, terwyl die vloeistof vrystellingsmetode van Bottrell en Yardley (1988) vir hierdie tegniek geoptimaliseer is. Kontaminasie faktore is geïdentifiseer en verminder of uitgeskakel waar moontlik. Daar is getoon dat die vloeistof chemie, wat verteenwoordigend is van al die insluitsel populasies in 'n monster, wel bruikbaar is t.o.v. afsonderlike vloeistof generasies, solank elke populasie individueel bestudeer is om die omvattende chemiese data te verduidelik en met 'n enkele populasie te korrelleer.

A series of rapid, high sensitivity capillary electrophoresis (CE) separation systems have been developed for the analysis of biological analytes and systems. A majority of the work has focused on development of novel instrumentation, in which new injection and detection strategies were investigated to improve the sensitivity of fast CE. A novel optical injection interface for capillary zone electrophoresis based upon the photophysical activation of caged dye attached to the target analyte was developed. The primary advantage of this approach is the lower background and background-associated noise resulting from reduced caged-fluorescein emission in conjunction with the high quantum yield of the resulting fluorescein. Improved detection limits were obtained compared to those observed in photobleaching-based optical gating. A primary drawback of photolytic optical gating CE is the lack of available caged-dye analogs with sufficiently fast reaction kinetics for online derivatization. To overcome this limitation, we have developed a chemical derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophysical properties of visible, high quantum yield, fluorescent dyes. The feasibility of this approach was evaluated by using an OPA/fluorescent thiol reaction, which was used to monitor neurotransmitter mixtures and proteins. The utilization of a high power ultraviolet light emitting diode for fluorescence detection in CE separations has been introduced to analyze a range of environmentally and biologically important compounds, including polyaromatic hydrocarbons and biogenic amines, such as neurotransmitters, amino acids and proteins, that have been derivatized with UV-excited fluorogenic labels. To understand cellular chemistry, it is imperative that single cells should be studied. This work was focused on developing CE based method to characterize the cellular uptake of TAT-EGFP. We demonstrated TAT mediated delivery of EGFP protein into HeLa cells and TAT-EGFP loaded single cell was analyzed by CE-LIF to determine the intracellular EGFP content. An application of CE-LIF for the determination of biogenic amine levels in the antennal lobes of the Manduca sexta is also explored and methods were developed to analyze a single antennal lobe dissected from moths. The lobe was digested and contents were labeled with the fluorogenic dye prior to CZE analysis.