<p>Suboptimal dosing of anti-microbial agents increases the likelihood of therapeutic failure and resistance. Dosing optimization, while an attractive approach to combat these issues, is difficult to implement due to the different pharmacokinetics of each individual. These limitations highlight the inadequacies of a “standardized” dosing strategy. Therapeutic drug monitoring (TDM) provides a tailored treatment for individuals while avoiding adverse side effects from compounds with a narrow therapeutic window where elevated concentrations of a drug cause organ toxicity. This strategy involves accurately measuring the concentration of the analyte and interpreting the results based on pharmacokinetic parameters. Clinicians then draw conclusions regarding dose adjustment for their patient. However, TDM is expensive and difficult to perform because measurements occur in biofluids. Rapid and robust methods are necessary to quantify antimicrobial agents at the institutional level to guide patient care toward improved outcomes in serious infection. Paper spray ionization (PS), an emerging ambient ionization technique for clinical settings, demonstrations a wide versatility both in analyte variety and applications. This technique offers a rapid, accurate method to analyze these compounds with low rates of false positives even when multiplexing.</p><p><br></p><p>The work herein explains the method development of assays for TDM of various antimicrobial agents. Chapters two and three describe ways to improve the quantitative capability of paper spray through substrate pre-treatment, modification, and manipulation of key factors. Chapter four describes real-world applications for paper spray utility in clinical settings with the cross-validation of antifungal agents against a “gold standard” method. The final chapter, while not clinical based, describes the method development process for a LC-MS/MS assay to detect urobilinoids in fly guts.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Suboptimal dosing of anti-microbial agents increases the likelihood of therapeutic failure and resistance. Dosing optimization, while an attractive approach to combat these issues, is difficult to implement due to the different pharmacokinetics of each individual. These limitations highlight the inadequacies of a “standardized” dosing strategy. Therapeutic drug monitoring (TDM) provides a tailored treatment for individuals while avoiding adverse side effects from compounds with a narrow therapeutic window where elevated concentrations of a drug cause organ toxicity. This strategy involves accurately measuring the concentration of the analyte and interpreting the results based on pharmacokinetic parameters. Clinicians then draw conclusions regarding dose adjustment for their patient. However, TDM is expensive and difficult to perform because measurements occur in biofluids. Rapid and robust methods are necessary to quantify antimicrobial agents at the institutional level to guide patient care toward improved outcomes in serious infection. Paper spray ionization (PS), an emerging ambient ionization technique for clinical settings, demonstrations a wide versatility both in analyte variety and applications. This technique offers a rapid, accurate method to analyze these compounds with low rates of false positives even when multiplexing. The work herein explains the method development of assays for TDM of various antimicrobial agents. Chapters two and three describe ways to improve the quantitative capability of paper spray through substrate pre-treatment, modification, and manipulation of key factors. Chapter four describes real-world applications for paper spray utility in clinical settings with the cross-validation of antifungal agents against a “gold standard” method. The final chapter, while not clinical based, describes the method development process for a LC-MS/MS assay to detect urobilinoids in fly guts.

Within the field of depression the “monoamine hypothesis” has been the leading theory to explain the biological basis of depression. This theory proposes that the biological basis of depression is due to a deficiency in one or more of three key neurotransmitter systems, namely noradrenaline, dopamine and serotonin which are thought to mediate the therapeutic actions of virtually every known antidepressant agent. Citalopram is a selective serotonin-reuptake inhibitor (SSRI) used for the treatment of depression and anxiety disorders. Citalopram is a racemic compound, in other words composed of a 50:50 mixture of two enantiomers (S-(+)-citalopram and R-(-)-citalopram) and with one of the enantiomers (S-(+)-citalopram) accounting for the inhibitory effect. At the time of introduction of citalopram the physician needed a therapeutic drug monitoring service to identify patients with interactions, compliance problems and for handling questions concerning polymorphic enzymes and drug metabolism. An achiral analytical separation method based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for routine therapeutic drug monitoring (TDM) of citalopram and its two main demethylated metabolites. As the data available on citalopram were from achiral concentration determinations and to be able to further investigate citalopram enantiomers effects and distribution, a chiral method for separation of the enantiomers of citalopram and its demethylated metabolites was established. The advances within chiral separation techniques have made measurement of the concentrations of the individual enantiomers in biological fluids possible. The process behind enantioselective separation is however not fully understood and the mechanism behind the separation can be further scrutinized by the use of multivariate methods. A study of the optimization and characterization of the separation of the enantiomers of citalopram, desmethylcitalopram and didesmethylcitalopram on an acetylated ß-cyclodextrin column, by use of two different chemometric programs - response surface modelling and sequential optimization was performed. Sequential optimization can be a quicker mean of optimizing a chromatographic separation; response surface modelling, in addition to enabling optimization of the chromatographic process, also serves as a tool for learning more about the separation mechanism. Studies of the antidepressant effect and pharmacokinetics of citalopram have been performed in adults, but the effects on children and adolescents have only been studied to a minor extent, despite the increasing use of citalopram in these age groups. A study was initiated to investigate adolescents treated for depression, with respect to the steady-state plasma concentrations of the enantiomers of citalopram and its demethylated metabolites. The ratios between the S- and R-enantiomers of citalopram and didesmethylcitalopram were in agreement with studies involving older patients. The concentrations of the S-(+)- and R-(-) enantiomers of citalopram and desmethylcitalopram were also in agreement with values from earlier studies. The results indicate that the use of oral contraceptives may have some influence on the metabolism of citalopram. This might be because of an interaction of the contraceptive hormones with the polymorphic CYP2C19 enzyme. Even though the SSRIs are considered less toxic compared with older monoamine-active drugs like the tricyclic/tetracyclic antidepressants, the risk of developing serious side effects such as ECG abnormalities and convulsions has been seen for citalopram, when larger doses have been ingested. Furthermore, fatal overdoses have been reported where citalopram alone was the cause of death. Data on the toxicity of each of the enantiomers in humans have not been reported and no data on blood levels of the enantiomers in cases of intoxication have been presented. An investigation was initiated on forensic autopsy cases where citalopram had been found at the routine screening and these cases were further analysed with enantioselective analysis to determine the blood concentrations of the enantiomers of citalopram and metabolites. Furthermore the genotyping regarding the polymorphic enzymes CYP2D6 and CYP2C19 were performed. In 53 autopsy cases, we found increasing S/R ratios with increasing concentrations of citalopram. We found also that high citalopram S/R ratio were associated with high parent drug to metabolite ratio and may be an indicator of recent intake. Only 3.8 % were found to be poor metabolizers regarding CYP2D6 and for CYP2C19 no poor metabolizer was found. Enantioselective analysis of citalopram and its metabolites can provide valuable information about the time that has elapsed between intake and death. Genotyping can be of help in specific cases but the possibility of pharmacokinetic interactions is apparently a far greater problem than genetic enzyme deficiency. / On the day of the public defence the status of article IV was: Submitted.

Magister Scientiae - MSc / Indinavir is a potent and well tolerated protease inhibitor drug used as a component of the highly active antiretroviral therapy (HAART) of HIV/AIDS, which results in pharmacokinetics that may be favourable or adverse. These drugs work by maintaining a plasma concentration that is sufficient to inhibit viral replication and thereby suppressing a patient’s viral load. A number of antiretroviral drugs, including indinavir, undergo metabolism that is catalysed by cytochrome P450-3A4 enzyme found in the human liver microsomes. The rate of drug metabolism influences a patient’s response to treatment as well as drug interactions that may lead to life-threatening toxic conditions, such as haemolytic anaemia, kidney failure and liver problems. Therapeutic drug monitoring (TDM) during HIV/AIDS treatment has been suggested to have a potential to reduce drug toxicity and optimise individual therapy. A fast and reliable detection technique, such as biosensing, is therefore necessary for the determination of a patient’s metabolic profile for indinavir and for appropriate dosing of the drugs. In this study biosensors developed for the determination of ARV drugs comprised of cysteamine self-assembled on a gold electrode, on which was attached 3-mercaptopropionic acid-capped palladium telluride (3-MPA-PdTe) or thioglycolic acid-capped palladium telluride (TGA-PdTe) quantum dots that are cross-linked to cytochrome P450-3A4 (CYP3A4) in the presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide. The quantum dots were synthesized in the presence of capping agents (3-MPA or TGA) to improve their stability, solubility and biocompatibility. The capping of PdTe quantum dots with TGA or 3-MPA was confirmed by FTIR, where the SH group absorption band disappeared from the spectra of 3-MPA-PdTe and TGA-PdTe. The particle size of the quantum dots (< 5 nm) was estimated from high resolution transmission electron microscopy (HRTEM) measurements. Optical properties of the materials were confirmed by UV-Vis spectrophotometry which produced absorption iii bands at ~320 nm that corresponded to energy band gap values of 3 eV (3.87 eV) for TGAPdTe (3-MPA-PdTe) quantum dots. The electrocatalytic properties of the quantum dots biosensor systems were studied by cyclic voltammetry (CV) for which the characteristic reduction peak at 0.75 V was used to detect the response of the biosensor to indinavir. Results for indinavir biosensor constructed with 3-MPA-SnSe quantum dots are also reported in this thesis. The three biosensors systems were very sensitive towards indinavir; and gave low limits of detection (LOD) values of 3.22, 4.3 and 6.2 ng/mL for 3-MPA-SnSe, 3-MPA-PdTe and TGA-PdTe quantum dots biosensors, respectively. The LOD values are within the ‘maximum plasma concentration’ (Cmax) value of indinavir (5 - 15 ng/mL) normally observed 8 h after drug intake.

>Magister Scientiae - MSc / The aim of this study was to compare the trough plasma levels of Aspen-Vancomycin® (AV); and Sandoz-Vancocin CP® (SV) in premature infants with suspected Methicillin Resistant Staphylococcus aureus (MRSA) infection. The study was designed as a prospective, double blind, randomised trial involving male and female premature infants admitted in the Neonatal Intensive care Unit (NICU) at Netcare Blaauwberg and N1-city Hospitals for treatment of suspected MRSA-infection between April 2012 and June 2013. The inclusion criteria were: 29-35 weeks postmenstrual age (PMA), informed and written consent from parents of each premature infant enrolled in the study. Blood samples (0.3-0.4ml) were collected for renal function test and vancomycin trough levels determination. Blood samples for vancomycin trough level assay were collected thirty minutes prior to the administration of the third dose of vancomycin. Statistical analysis was performed and estimation was made giving an indication of how many infants will be needed to make the study statistically significant. Wilcoxon Two-Sample test was performed to determine the p-values and Spearman correlation coefficients were used to determine the correlation between trough levels and variables. P-values < 0.05 were considered significant. A total of 19 premature infants met with study criteria, 10 (5 females and 5 males) received AV and 9 (6 females and 3 males) receive d SV. There was no statistical significant difference between the demographic (GA, BW, PMA, PNA, weight at trial entry, height at trial entry) and biological (albumin, serum creatinine concentration and glomerular filtration rate) parameters of the premature infants in the AV and SV group. There were no statistical significant difference between trough level 1 of AV and SV, although trough level 1 had a lower trend in the SV group (p=0.118). No AV trough level 1 was below the minimum effective concentration (<5μg/ml). It was found that 30% of AV trough level 1 was within the therapeutic range (5-10μg/ml) and 70% of AV trough level 1, were above minimum toxic concentration (>10mg/l). It was found that 22.2% of SV trough level 1 was below minimum effective concentration, 44.4% of SV trough level 1 was within therapeutic range and 33.3% of trough level 1 was above minimum toxic concentration. No correlation was found between trough level 1 and the demographic and biological parameters of the premature infants in the AV group. SV had a positive correlation with GA, BBW, PMA and a negative correlation with PNA