@phdthesis{Schilling2020,
  author    = {Schilling, Klaus Jussi},
  title     = {Liquid chromatographic analysis of weakly- and non-chromophore compounds focusing on Charged Aerosol Detection},
  doi       = {10.25972/OPUS-20211},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202114},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Liquid chromatography has become the gold standard for modern quality control and purity analytics since its establishment in the 1930s. However, some analytical questions remain very challenging even today. Several molecules and impurities do not possess a suitable chromophore for the application of UV detection or cannot be retained well on regular RP columns. Possible solutions are found in derivatization procedures, but they are time consuming and can be prone to errors. In order to detect non chromophore molecules underivatized, the concept of aerosol based universal detection was established with the introduction of the evaporative light scattering detector (ELSD) in the 1970s and the charged aerosol detector (CAD) followed in 2002. These two challenging fields - polar and non chromophore molecules - are tackled in this thesis. An overview of applications of the CAD in the literature and a comparison to its aerosol based competitors and MS is presented, emphasizing on its high sensitivity and robustness. Parameters and techniques to overcome the drawbacks of CAD, such as the use of gradient compensation or adjusted evaporation temperatures are discussed. A consideration of aspects and drawbacks of data transformation such as the integrated power function value (PFV) in the GMP environment is performed. A method for the fatty acid analysis in polysorbate 80 that was developed on HPLC CAD was transferred to UHPLC CAD. Time and eluent savings of over 75\% and 40\%, respectively, as well as ways to determine the optimal CAD parameters resulted from this investigation. The evaporation temperature was determined as the most crucial setting, which has to be adjusted with care. Optimal signal to noise ratios are found at a compromise between maintaining analyte signal and reducing background noise. The incorporation of semi volatile short chain fatty acids enabled the observation of differences based on volatility of the analyte. E.g. for semi volatiles, an improved linearity by means of adjusting the PFV is achieved at values below 1.0 instead of at elevated PFVs. Using sugars and sugar related antibiotics, a proof-of-concept was given that artificial neural networks can describe correlations between the structure and physicochemical properties of molecules and their response in CAD. Quantitative structure property relationships obtained by design of experiment approaches were able to predict the response of unseen substances and yielded insights on the response generation of the detector, which heavily relies on the formed surface area of the dried particle. Further work can substantiate upon these findings, eventually building a library of diverse eluent compositions, analytes and settings. In order to cope with a chromatographically challenging substances, the application of ion pairing reversed phase chromatography coupled to low wavelength UV detection has been shown as a possible approach for the amino acid L asparagine. A method capable of compendial purity analysis in one single HPLC approach, thus making the utilization of the semi quantitative TLC-ninhydrin analysis obsolete, resulted from this. One cyclic dipeptide impurity (diketoasparagine) that was formerly not assessed, could be identified in several batches and added to the monograph of the Ph.Eur. Studying ibandronate sodium with CAD and ELSD, it was found that randomly occurring spike peaks represent a major flaw of the ELSD when high sample load is present. The research with this non chromophore bisphosphonate drug furthermore shed light on possible drawbacks of mixed mode chromatography methods and ways to overcome these issues. Due to strong adsorption of the analyte onto the column, over ten injections of the highly concentrated test solution were found to be necessary to ensure reproducible peak areas. Preconditioning steps should thus be evaluated for mixed mode approaches during method development and validation. Last, using a ternary mixed mode stationary phase coupled to CAD, a method for the impurity profiling of pamidronate disodium, also applicable to the assessment of phosphate and phosphite in four other bisphosphonate drugs, has been developed. This represents a major advantage over the Ph.Eur. impurity profiling of pamidronate, which requires two different methods, one of which is only a semi quantitative TLC approach.},
  subject      = {HPLC},
  language  = {en}
}
@phdthesis{Reggane2019,
  author    = {Reggane, Maude},
  title     = {Lowering lattice forces of crystalline bases},
  doi       = {10.25972/OPUS-16380},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163803},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The number of active pharmaceutical ingredients (APIs) exhibiting a low solubility in aqueous media or a slow dissolution rate kept rising over the past years urging formulation scientists to explore new ways to tackle poor solubility and to enable oral absorption from such compounds. Bioavailability of poorly water-soluble compounds can be improved by increasing the dissolution rate and/or by increasing the gastro intestinal concentration through transient supersaturation. The dissolution rate of the API can be typically modified by the choice of the physical form, the polymorphic form, the powder surface area, and the local pH, while a transient supersaturation can be extended mainly by nucleation or crystallization inhibiting effects. In the present thesis, three strategies were explored to tailor the dissolution rate, the supersaturation and the hydrotropic solubilization of APIs, weak bases, respectively. The first part of this thesis followed a bioinspired approach to extend the kinetic solubility of salts and co-crystals. API salts and co-crystals are high energy forms that can generate supersaturated solutions with respect to any more stable form, typically the most stable API form in physiological environment. The transient kinetic stabilization of supersaturated states, also termed "parachute effect", is considered to improve bioavailability and is one aspect of the formulation that can be tailored. Inspiration from plants, which store high concentrations of aromatic bases in their vacuoles via complexation with polyphenols, sparked the evaluation to use hydroxybenzoic acid derivatives for salt or co-crystal engineering. Imatinib was chosen as the model compound for this investigation as its aromaticity and flat molecular architecture could favor interactions with hydroxybenzoic acid derivatives. One 1:1 Imatinib syringate co-crystal (I-SYA (1:1)) and one 1:2 Imatinib syringate co-crystal salt (I-SYA (1:2)) were obtained. Their dissolution assays in simulated intestinal fluid (SIF; a 50 mM phosphate buffer of pH 6.8) revealed that they formed stable solutions for several hours and days, respectively, in contrast to the marketed Imatinib mesylate salt (approx. 1h). This kinetic stability in solution was linked to the nucleation inhibition of the less soluble Imatinib hydrate by syringic acid (SYA). In solution 1H-NMR studies evidenced the aggregation of Imatinib and SYA. The amphiphilic nature of both Imatinib and SYA is considered to drive their association in solution, additionally, multiple intermolecular interactions such as hydrogen bonds and π-π stacking are likely to contribute. The association in solution enabled a phase of extended supersaturation, i.e., a parachute against desupersaturation, while no negative impact of aggregation on the permeability of both Imatinib and SYA was observed. A prerequisite to reach supersaturation is a rapid dissolution and release of the API from the formulation. Accordingly, the second and third part of this thesis is focused on the so-called "spring effect" of amorphous solid dispersions (ASDs). The addition of a hydrotropic agent, meaning a molecule that can solubilize poorly water-soluble APIs in aqueous solutions (well-known examples of hydrotropes are benzoic acid and nicotinamide) into an amorphous Ciprofloxacin-polymer matrix led to ternary systems with a significantly faster release and higher concentration of the API in SIF as compared to binary ASDs consisting of Ciprofloxacin (CPX) and polymer only. The stronger spring could be rationalized by an improved wetting of the ASD, or/and by a hydrotropic solubilization effect, although these hypotheses need further investigation. Marked differences in the dissolution profiles of binary ASDs were observed in biorelevant fasted simulated intestinal fluid (FaSSIF; a medium containing Na taurocholate (3 mM) and lecithin (0.75 mM) at pH 6.5) as compared to SIF. In FaSSIF, API release from binary polymeric ASDs was largely improved, and the duration of supersaturation was extended. This suggests that the bile salt Na taurocholate and lecithin present in FaSSIF do improve both dissolution rate and supersaturation of ASDs, the two pillars of ASDs as oral enabling formulations. Indeed, bile salts are endogenous surfactants which, together with phospholipids, play an important role in the wetting, solubilization, and absorption of lipophilic compounds. The aim of the third part of the present thesis was to study ASDs as formulation principles reducing the strong positive food effect of Compound A. By inclusion of Na taurocholate (NaTC) within the matrix of polymeric ASDs a significant improvement of the dissolution rate and the kinetic solubility in SIF were achieved. Transient supersaturated states of up to four orders of magnitude over the equilibrium solubility were obtained. Two ASDs were selected for further in vivo evaluation in dog. The first was a NaTC/Eudragit E based ASD meant to dissolve and release Compound A in the acidic environment of the stomach, where its solubility is the highest. The second relied on the release of Compound A in the neutral environment of the duodenum and jejunum by using an enterically dissolving polymer, HPMC-P. Releasing the API at the site of its putative absorption was an attempt to control supersaturation levels in the duodenum and to prevent portioning and thus dilution effects during transfer from the stomach. In fasted dogs, exposure from the NaTC/HPMC-P ASD was close to that of the reference Compound A formulation under fed conditions, which suggests an improved dissolution rate and kinetic solubility under fasted conditions (historical data). The exposure from the NaTC/Eudragit E ASD was twice as low as from the NaTC/HPMC-P ASD, and also lower compared to Compound A reference formulation, whereas in vitro the parachute effect of the NaTC/Eudragit E ASD was largely superior to that of the NaTC/HPMC-P ASD. A difference in the extend of the parachute could be related to differences in the thermodynamic activity of dissolved molecules from the two ASDs. Indeed, the high instability of the NaTC/HPMC-P ASD could stem from a high thermodynamic activity driving diffusion through membranes, whereas less instable solutions of NaTC/Eudragit E could indicate solubilization effects which often translate into a lower flux through the biological membrane. Additionally, the pH of the environment where dissolution takes place might be an important factor for absorption, and could also account for the difference in exposure from the two ASDs. The aim of this thesis was to explore how the intimate environment of weak, poorly soluble bases could be functionalized to improve dissolution rate and kinetic solubility. The investigations highlighted that the performance of enabling oral delivery formulations of weak bases in aqueous media can be enhanced at different levels. At one end initial dissolution rate of ASDs can be tailored by introducing hydrotropes or/and bile salts within the polymeric matrix of ASDs. Bile salts, when combined with appropriate polymers, had also a precipitation inhibition effect enabling the maintenance of supersaturation for a bio-relevant period of time. These results set the ground for further investigations to comprehend specific interactions between bile salts and APIs, and potentially polymers at the molecular level. It will be interesting to explore how such complex systems can be exploited in the formulation design of poorly water-soluble APIs. In addition, it was observed that the duration of supersaturation generated by salts/co-crystals can be extended by the pertinent selection of counterions or coformers. The in vivo relevance of these tunings remains to be evaluated, as translation from closed, in vitro systems to the highly dynamic gastrointestinal environment is not straightforward. A better understanding of the contribution of each kinetic stage (dissolution, supersaturation, and precipitation) and their interplay with physiological factors impacting absorption is essential to facilitate the design of formulations with improved pharmacokinetics.},
  subject      = {Kokristallisation},
  language  = {en}
}
@phdthesis{SchuesslergebHecht2018,
  author    = {Sch{\"u}ßler [geb. Hecht], Nina Kristin Petra},
  title     = {Novel formulation principles for bioavailability enhancement of poorly water-soluble and poorly permeable drugs},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162766},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Since four decades, high-throughput screenings have been conducted in drug discovery, fuelling the identification of potential new drug candidates. This approach, however, often promotes the detection of compounds with undesired physico-chemical properties like poor aqueous solubility or low membrane permeability. Indeed, dissolution and absorption of a drug are prerequisites for systemic exposure and therapeutic effects. Therefore, innovative strategies to optimize unfavourable performance of new drug candidates are in great demand in order to increase drug concentrations at the site of action whilst simultaneously reducing drug variability. In chapter I of this research work, hydrophobic ion pairing (HIP) is discussed as a promising strategy to improve the bioavailability of BCS class III compounds, which have high aqueous solubility and low permeability. The review points out the limitations of poorly absorbable drugs and details the approach of pairing these APIs with hydrophobic counterions. Apart from the motivation to tailor physico-chemical, biopharmaceutical and toxicological properties of BCS class III compounds, the hydrophobic ion pairing facilitates their formulation into drug delivery systems. Besides advantageous effects, disadvantages of the ion pair formation, such as the decreased aqueous solubility of the ions pair, are critically outlined. Finally, the review covers an overview of non-invasive administration routes permitted after ion pair formation, including oral/enteral, buccal, nasal, ocular and transdermal drug administration. Overall, the HIP approach offers substantial benefits regarding the bioavailability enhancement of BCS class III compounds. Chapter II concerns GHQ168 developed by Holzgrabe et al., a BCS class II compound characterized by low aqueous solubility and high permeability. GHQ168 was developed for the treatment of human African trypanosomiasis (HAT), a tropical disease for which novel active compounds are urgently needed. This lead compound was found to be very active against trypanosoma brucei brucei and trypanosoma brucei rhodesiense in cell culture assays, however, the low aqueous solubility prevented further preclinical development. To target this drawback, two different approaches were selected, including (I) the chemical modification and (II) the spray drying of GHQ168. The newly synthesized set of derivatives as well as the spray dried GHQ168 were subjected to a physico-chemical and microbiological characterization. It turned out that both approaches successfully improved aqueous solubility, however, for the derivatives of GHQ168 at the expense of activity. Furthermore, the pharmacokinetic parameters of GHQ168 and of the most active derivatives, GHQ242 and GHQ243, were evaluated. Elimination half-lives between 1.5 to 3.5 h after intraperitoneal administration and modest to strong serum albumin binding for GHQ243 (45\%) and GHQ168 (80\%) and very high binding (> 99\%) for GHQ242 were detected. The spray dried formulation of GHQ168, as well as GHQ242 and GHQ243 were investigated in two in vivo studies in mice infected with t. b. rhodesiense (STIB900), referred to as (I) stringent model and (II) early-treatment model. In the stringent model (2 applications/day on day 3-6 after infection) the mean survival duration (MSD) of mice treated with spray dried GHQ168 exceeded the MSD of the untreated control group (17 days versus 9 days), a difference that was statistically significant. In contrast, no statistical difference was observed for GHQ242 (14 days) and GHQ243 (12 days). GHQ168 was further assessed in the early-treatment model (2 applications/day on day 1-4 after infection) and again a statistically significant improvement of MSD (32 days (end of observation period) versus 7 days) was observed. Finally, exciting antitrypanosomal efficacy for the spray dried formulation of GHQ168 was demonstrated. NADPH oxidases (NOX) were found to be the main source of endothelial reactive oxygen species (ROS) formation. Chapter III reports on the formulation studies on triazolopyrimidine derivatives from the VAS library, a set of NADPH oxidase inhibitors. These were developed for the treatment of elevated ROS levels, which contribute to the development of cardiovascular diseases. Although in vitro results from numerous studies indicated promising efficacy and selectivity for the VAS-compounds, the low water solubility impeded the in vivo translation and further preclinical development. For this reason, three derivatives, VAS2870, VAS3947, and VAS4024 were physico-chemically characterized and VAS3947, the most soluble compound, was selected for further formulation studies. These approaches included (I) spray drying, (II) microemulsification and (III) complexation with cyclodextrins in order to develop formulations for oral and parenteral application. Solubility improvement of VAS3947 was successfully demonstrated for all preparations as expressed by supersaturation ratios in comparison to the solubility of the unformulated compound. For seven spray dried formulations, the ratio ranged from 3-9, and the ratio for four microemulsions was 8-19 after 120 min, respectively. The six cyclodextrin formulations achieved the highest supersaturation ratio between 3 and 174 after 20 hours. NMR measurements elucidated the inclusion of VAS3947 within the CD's cavity as well as the interaction with its outer surface. Ultimately, NOX inhibitors were opened for oral and parenteral administration for the first time. After successful solubility improvement of VAS3947, further investigations towards in vivo studies were conducted including stability studies with a focus on stability in solution and in plasma as presented in chapter IV. Furthermore, permeability and cytotoxicity assays were performed for the first time. It turned out that VAS3947 was instable in buffer and when exposed to light. Moreover, the compound showed decomposition in the presence of mouse microsomes and in human plasma. The VAS compounds contain an oxazol moiety linked to the triazolopyrimidine skeleton via a thioether. This structural element is responsible for the efficacy of the compound class, however it is susceptible to hydrolysis and to further degradation reactions. Moreover, VAS3947 harmed membrane integrity in the cell permeability assays and cytotoxicity investigations in HEK-293 and HEP-G2 cells revealed IC50 values in the same concentration range as reported for efficacy assays. Summarized, it was demonstrated that substances from the VAS library were no appropriate model compounds for ROS investigations nor suitable candidates for further preclinical development.},
  subject      = {L{\"o}slichkeit},
  language  = {en}
}
@phdthesis{Goetz2019,
  author    = {G{\"o}tz, Marcus Rudolf},
  title     = {Effiziente Synthese von Dronabinol und weiterer cannabinoider Derivate und deren pharmakologische Charakterisierung},
  doi       = {10.25972/OPUS-16662},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-166625},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In dieser Arbeit wurde ein Verfahren zur effizienten Herstellung von (-)-trans-Cannabidiol (CBD, 10), (-)-trans-Δ9-Tetrahydrocannabinol (Dronabinol, 21) und (-)-trans-Cannabidivarin (CBDV, 30) durch kontinuierliche Synthese untersucht und entwickelt. CBD konnte durch kontinuierliche Synthese in drei Schritten aus Olivetolcarbons{\"a}uremethylester (OM, 6) und Menthadienol G (3) mit einer Ausbeute von 41 \% synthetisiert werden. Bei optimierten Bedingungen betrug die Reinheit nach Kristallisation > 99 \%. Die Stereochemie konnte durch R{\"o}ntgenstrukturanalyse eindeutig als 1R,6R bestimmt werden. Vorteilhaft war dabei, dass Toluol anstatt eines chlorierten L{\"o}sungsmittels verwendet werden konnte. Weitere Vorteile waren die kurze Reaktionszeit und die Tatsache, dass die Synthese bei Raumtemperatur durchgef{\"u}hrt werden konnte. Es konnten f{\"u}nf Nebenprodukte detektiert und identifiziert werden, wovon eines Dronabinol war. Bei optimierten Reaktionsparametern konnte eine Ausbeute an Dronabinol von 64,5 \% erreicht werden. Durch Simulated Moving Bed (SMB)-Chromatographie konnte Dronabinol kontinuierlich mit einem Gehalt von > 95 \% hergestellt werden. Nach der Synthese waren vier Verunreinigungen detektierbar, und zwar Olivetol (17), CBD, Exo-Tetrahydrocannabinol (Exo-THC, 23) und Δ8-Tetrahydrocannabinol (Δ8-THC, 22). Durch die SMB-Aufreinigung konnten alle Verunreinigungen auf einen monographiekonformen (USP 37) Gehalt abgereichert werden. Nach der finalen destillativen Aufarbeitung trat eine noch nicht identifizierte Verunreinigung in einem Gehalt von ca. 0,4 Fl{\"a}chen-\% auf. CBDV konnte durch kontinuierliche Synthese in drei Schritten aus Divarincarbons{\"a}uremethylester (DM, 25) und Menthadienol G synthetisiert werden. Die Ausbeute betrug ca. 30 \%, die Reinheit nach Kristallisation > 99 \%. Es konnten f{\"u}nf Nebenprodukte detektiert werden, die im Rahmen dieser Arbeit nicht weiter charakterisiert wurden. Der Syntheseweg bietet durch Modifikation der Seitengruppen an Position 6 (R1) und Position 5 (R2) der Alkylbenzol-Gruppe Zugang zu synthetischen Cannabinoiden mit einem CBD- oder CBDV-Grundger{\"u}st. Es wurden neun neue Cannabinoide hergestellt: 2-Hydroxyethylcannabidiolat (2-HEC, 31), 2-Hydroxypentylcannabidiolat (2 HPC, 32), Glycerylcannabidiolat (GCBD, 33), Cyclohexylcannabidiolat (CHC, 34), Hexylcannabidiolat (HC, 35), N-Methylsulfonylcannabidiolat (NMSC, 36), 2 Hydroxyethylcannabidivarinolat (2-HECBDV, 37), Cyclohexylcannabidivarinolat (CHCBDV, 38) und Hexylcannabidivarinolat (HCBDV, 39). Die Bindungsaffinit{\"a}t wurde in Cannabinoid-Rezeptor-transfizierten HEK293EBNA-Zellen untersucht, die intrinsische Aktivit{\"a}t in CHO-Zellen, die Induktion von NF-κB (nuclear factor kappa B) sowie von NFAT (nuclear factor of activated T cells) in Jurkat-T Zellen, die Induktion proinflammatorischer Zytokine und Chemokine (Interleukin(IL)-6, IL-1β, CC Chemokinligand 2' (CCL2) und Tumornekrosefaktor(TNF)-α) auf mRNA-Ebene in RAW264.7-Makrophagen und die Expression von proinflammatorischen Zytokinen (IL-1β, IL-6, IL-8, TNF-α) und Prostaglandin E2 (PGE2) auf Proteinebene in prim{\"a}ren humanen Monozyten. Die CBD-Derivate zeigten eine h{\"o}here Selektivit{\"a}t f{\"u}r CB2-Rezeptoren. Die CBDV-Derivate HCBDV und CHCBDV zeigten eine spezifische Bindung an CB1- und CB2-Rezeptoren im nanomolaren Bereich. 2-HEC, 2-HPC, GCBD und NMSC wirkten als Agonisten an CB2- und als Antagonisten am CB1-Rezeptor. CHC band an CB1 und CB2 im submikromolaren Bereich und schien ein Agonist f{\"u}r beide Rezeptoren zu sein. 2- HECBD wirkte als Agonist auf CB2-Rezeptoren und als Antagonist auf CB1-Rezeptoren. In Jurkat-T Zellen hemmte NMSC dosisabh{\"a}ngig die Aktivit{\"a}t von NF-κB sowie von NFAT. 2-HEC, 2-HPC und GCBD hemmten die Expression von NFAT ebenfalls dosisabh{\"a}ngig. CHC und HC reduzierten dosisabh{\"a}ngig die Expression von IL-1β- und CCL2-mRNA in RAW264.7-Makrophagen. NMSC hemmte in geringeren Dosen IL-1β, CCL2 sowie TNF-α und induzierte in h{\"o}heren Dosen einen starken Anstieg der IL-6-mRNA. In prim{\"a}ren humanen Monozyten hemmten 2 HEC und GCBD konzentrationsabh{\"a}ngig die Synthese von IL-1β, IL-6 und TNF-α. 2-HPC hemmte dosisabh{\"a}ngig die Bildung von TNF-α und IL-6. HC verminderte dosisabh{\"a}ngig die Freisetzung von TNF-α und IL-6. NMSC steigerte die durch LPS erh{\"o}hte Freisetzung von IL-1β noch weiter, hemmte aber TNF-α, IL-8 und PGE2. Die hier untersuchten CBD- und CBDV-Derivate sind geeignet, gezielt an Cannabinoid-Rezeptoren zu wirken. Einige der Derivate k{\"o}nnten als selektive CB2-Agonisten genutzt werden. Die L{\"a}nge des aliphatischen Rests an R2 von CBD (Pentyl-Cannabinoiden) und CBDV (Propyl-Cannabinoiden) korrelierte nicht mit der Bindungsaffinit{\"a}t. Eine h{\"o}here Polarit{\"a}t an R1 (2-HECBDV > NMSC > GCBD > 2-HEC) schien demgegen{\"u}ber die agonistische Aktivit{\"a}t an CB2 zu beg{\"u}nstigen. Um den Ergebnissen zur Beziehung zwischen Struktur und Wirkung noch mehr Bedeutung zu geben, w{\"a}ren weitere synthetische Derivate und deren Testung notwendig.},
  subject      = {Dronabinol},
  language  = {de}
}
@phdthesis{Wahl2019,
  author    = {Wahl, Joachim},
  title     = {The Use of Ionic Liquids in Capillary Electrophoresis Enantioseparation},
  doi       = {10.25972/OPUS-17639},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176397},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Two chiral chemical molecules being mirror images of each other, also referred to as enantiomers, may have different pharmacokinetic, pharmacodynamic, and toxicological effects. Thus, pharmaceutical manufacturers and authorities are increasingly interested in the approval of enantiopure drugs. However, the isomeric purity and the limits for isomeric impurities have to be specified applying enantioselective analytical methods, such as capillary electrophoresis. The separation of enantiomers in capillary electrophoresis may be improved by the addition of ionic liquids to the background electrolyte. The aim of this work was to investigate the influence of different separation conditions on the enantioseparation of phenethylamines in background electrolytes containing ionic liquids based on tetrabutylammonium cations. Best chiral separations were achieved at acidic pH values using phosphate buffers containing 125 mmol/L tetrabutylammonium based salts. Different reasons explaining enhanced enantioseparations in buffers containing ionic liquids were found. First, due to an improvement of the cyclodextrin solubility, the addition of ionic liquids to the background electrolyte enables the use of higher concentrations of these chiral selector. Furthermore, the adsorption of tetrabutylammonium cations to the negatively charged capillary surface results in a reduction of the electroosmotic flow. Hence, the resulting prolongation of migration times leads to a longer period of time for the separation of temporarily formed diastereomeric analyte cyclodextrin complexes, which yields improved enantioseparation. Additionally, due to a decrease of the adsorption of positively charged phenethylamine analyte molecules to capillary surface silanol groups, the adsorption of ionic liquid cations inhibits peak broadening. A further reason explaining an enhanced enantioseparation by the addition of ionic liquids to the background electrolyte is a competition between tetrabutylammonium cations and analyte enantiomers for the inclusion into cyclodextrin cavities. Furthermore, the influence of different chiral counterions, combined with tetrabutylammonium cations, on the enantioseparation of phenethylamines was investigated. Solely anions based on the basic proteinogenic amino acids L lysine and L arginine yielded chiral separation results superior to those achieved using achiral tetrabutylammonium chloride as background electrolyte additive. Especially the application of tetrabutylammonium L argininate gave very good enantioseparations of all investigated ephedrine derivatives, which might be explained by the ability of L arginine to affect the formation of complexes between analytes and cyclodextrins. Besides the investigation of the influence of ionic liquids on the enantioseparation, complexes between phenethylamine enantiomers and β cyclodextrin derivatives were characterized by affinity capillary electrophoresis. The binding constants between analyte enantiomers and cyclodextrins and the electrophoretic mobilities of the temporarily formed complexes were determined and compared to the observed chiral resolution values. While neither the calculated binding constants nor their differences correlated with the quality of the enantioseparation, a strong correlation between the differences of the electrophoretic mobilities of the complexes and the chiral resolution values was found.},
  subject      = {Kapillarelektrophorese},
  language  = {en}
}
@phdthesis{Berninger2019,
  author    = {Berninger, Michael},
  title     = {Development of Novel Quinolone Amides Against the African Sleeping Sickness - A Fluorine Walk},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176428},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In recent years the transmission of the Human African Trypanosomiasis could be significantly reduced. The reported cases in 2016 reached a historic low level of 2184 cases and these achievements can be ascribed to intense control and surveillance programmes.118 However, most of the reported cases (>1000 in 2015) occurred in the Democratic Republic of the Congo and thus, need to be treated adequately. In particular, when the parasites have traversed the blood-brain barrier (BBB), treatment proved to be even more difficult. In addition, the number of cases always came in waves due to many reasons, e.g., development of resistances. Thus, it can be expected from experiences of the past that the number of cases will increase again. Hence, novel chemical entities are desperately needed in order to overcome the drawbacks which are associated with the current treatment options. Our drug discovery approach included an initial drug repurposing strategy combined with a phenotypic screening. S. Niedermeier found novel active compounds derived from commercial fluoroquinolones. The most promising hit compound was further developed by G. Hiltensperger resulting in the lead quinolone amide GHQ168 (IC50 = 0.047 µM). This doctoral thesis is about new insights into the SAR of the quinolone amides and the enhancement of the lead compound. Special consideration was given to the fluorine atom in the quinolone amides and how certain fluorine substitution patterns influence the antitrypanosomal activity, physicochemical properties and pharmacokinetics (i.e. 'fluorine walk'). Moreover, the ability of the compound class crossing the BBB should be investigated. This feature is inevitable necessary in order to potentially treat African sleeping sickness stage II. The Gould-Jacobs protocol was predominantly used for the synthesis of the quinolone core. Since former SAR studies mainly concentrated on the variation in positions 1, 3 and 7, quinolone scaffolds (2a-i) with diverse substitution patterns regarding positions 5, 6, 7 and 8 were synthesised in this thesis. The resulting quinolone amides were evaluated for their antitrypanosomal activity. Voluminous residues in position C-5 resulted in diminished activities (compounds 13, 16 and 18) and solely small-sized moieties were tolerated. In particular the fluorine atom in position 5 revealed beneficial trypanocidal effects as shown for compounds 6 (IC50 = 0.05 µM), 8 (IC50 = 0.04 µM), and 24 (IC50 = 0.02 µM). Furthermore, having fluorine only in position 5 of the quinolone core could considerably reduce the cytotoxic effects (CC50 >100 µM, SI = >2000 for 6). Hence, the 5-fluoro-substituted quinolone amides were considered superior to GHQ168. Regarding the C-6 position all other moieties (e.g., H in 9, OCH3 in 10, CF3 in 12) except of a fluorine atom decreased the activity against Trypanosoma brucei brucei. A double fluorination in C-6 and C-8 was not beneficial (IC50 = 0.06 µM for 7) and a single fluorine atom in C-8 even showed a negative effect (IC50 = 0.79 µM for 5). The logP value is considered a surrogate parameter for lipophlicity and thus, affecting permeability and solubility processes. In particular the fluorine atom influences the lipophilicity due to versatile effects: Lipophilicity is increased by additional fluorine atoms on aromatic rings (7, 23) and reduced by fluorine atoms at an alkyl chain (49), respectively. Additionally, the 5-fluoro-substituted quinolone amides (6, 8, and 24) could prove the contrary effect of decreasing lipophilicity when the aromatic fluorine substituent is in vicinity to a carbonyl group. For the most promising drug candidates 6, 23, and 24 the respective metabolites and the metabolic turnover were investigated by C. Erk. In comparison to GHQ168 the hydroxylation of the benzylamide was prevented by the para-fluorine atom. Hence, half-life was extended for compound 23 (t1/2 = 6.4 h) and N-desalkylation was the predominant pathway. Moreover, the respective fluorine substitution pattern of the quinolone core affected the metabolism of compound 6. The 5-fluoro-substituted quinolone amide was less prone for biotransformation (t1/2 = 7.2 h) and half-life could even be further prolonged for compound 24 (t1/2 = 7.7 h). Due to the most appropriate safety profile of compound 6, this particular drug candidate was considered for in vivo study. Its poor solubility made a direct intraperitoneal administration unfeasible. Thus, an amorphous solid dispersion of 6 was generated using the spray-drying method according to the previous protocol. Unfortunately, the required solubility for the predicted in vivo study was not achieved. Furthermore, the compound class of the quinolone amide was evaluated for its ability for brain penetration. The methanesulfonyl precursor 48 was synthesised and subsequently radiofluorinated in the group of Prof. Dr. Samnick (Department of Nuclear Medicine, University Hospital of W{\"u}rzburg). The labelled compound [18F]49 was administered to mice, and its distribution throughout the body was analysed using positron emission tomography and autoradiography, respectively. The autoradiography of the murine brains revealed medium to high concentrations of [18F]49. Therefore, the quinolone amides are generally suitable for treating Human African Trypanosomiasis stage II. A scaffold hopping approach was performed starting from the quinolone amides and concluding with the compound class of pyrazoloquinolin-3-ones. The intramolecular hydrogen bond between the sec. amide and the C-4 carbonyl moiety was replaced by a covalent bond. The two compound classes were comparable regarding the antitrypanosomal activity to some degree (IC50 = 7.9 µM (EK02) vs. 6.37 µM (53a)). However, a final evaluation of 59 was not possible due to poor solubility.},
  subject      = {Trypanosomiase},
  language  = {en}
}
@phdthesis{Mohsen2017,
  author    = {Mohsen, Amal Mahmoud Yassin},
  title     = {Structure Activity Relationships of Monomeric and Dimeric Strychnine Analogs as Ligands Targeting Glycine Receptors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142228},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The inhibitory glycine receptors are one of the major mediators of rapid synaptic inhibition in the mammalian brainstem, spinal cord and higher brain centres. They are ligand-gated ion channels that are mainly involved in the regulation of motor functions. Dysfunction of the receptor is associated with motor disorders such as hypereklepxia or some forms of spasticity. GlyR is composed of two glycosylated integral membrane proteins α and β and a peripheral membrane protein of gephyrin. Moreover, there are four known isoforms of the α-subunit (α1-4) of GlyR while there is a single β-subunit. Glycine receptors can be homomeric including α subunits only or heteromeric containing both α and β subunits. To date, strychnine is the ligand that has the highest affinity as glycine receptor ligand. It acts as a competitive antagonist of glycine that results in the inhibition of Cl- ions permeation and consequently reducing GlyR-mediated inhibition. For a long time, the details of the molecular mechanism of GlyRs inactivation by strychnine were insufficient due to the lack of high-resolution structures of the receptor. Only homology models based on structures of other cys-loop receptors have been available. Recently, 3.0 {\AA} X-ray structure of the human glycine receptor- α3 homopentamer in complex with strychnine, as well as electro cryo-microscopy structures of the zebra fish α1 GlyR in complex with strychnine and glycine were published. Such information provided detailed insight into the molecular recognition of agonists and antagonists and mechanisms of GlyR activation and inactivation. Very recently, a series of dimeric strychnine analogs obtained by diamide formation of two molecules of 2-aminostrychnine with diacids of different chain length was pharmacologically evaluated at human α1 and α1β glycine receptors. None of the dimeric analogs was superior to strychnine. The present work focused on the extension of the structure-activity relationships of strychnine derivatives at glycine receptors All the synthesized compounds were pharmacologically evaluated at human α1 and α1β glycine receptors in a functional FLIPRTM assay and the most potent analogs were pharmacologically evaluated in a whole cell patch-clamp assay and in [3H]strychnine binding studies. It was reported that 11-(E)-isonitrosostrychnine displayed a 2-times increased binding to both α1 and α1β glycine receptors which prompted us to choose the hydroxyl group as a suitable attachment point to connect two 11-(E)-isonitrosostrychnine molecules using a spacer. In order to explore the GlyR pocket tolerance for oxime extension, a series of oxime ethers with different spacer lengths and sterical/lipophilic properties were synthesized biologically evaluated. Among all the oxime ethers, methyl, allyl and propagyl oxime ethers were the most potent antagonists displaying IC50 values similar to that of strychnine. These findings indicated that strychnine binding site at GlyRs comprises an additional small lipophilic pocket located in close proximity to C11 of strychnine and the groups best accommodated in this pocket are (E)-allyl and (E)-propagyl oxime ethers. Moreover, 11-aminostrychnine, and the corresponding propionamide were prepared and pharmacologically evaluated to examine the amide function at C11 as potential linker. A series of dimeric strychnine analogs designed by linking two strychnine molecules through amino groups in position 11 with diacids were synthesized and tested in binding studies and functional assays at human α1 and α1β glycine receptors. The synthesized bivalent ligands were designed to bind simultaneously to two α-subunits of the pentameric glycine receptors causing a possibly stronger inhibition than the monomeric strychnine. However, all the bivalent derivatives showed no significant difference in potency compared to strychnine. When comparing the reference monomeric propionamide containing ethylene spacer to the dimeric ligand containing butylene spacer, a 3-fold increase in potency was observed. Since the dimer containing (CH2)10 spacer length was found to be equipotent to strychnine, it is assumed that one molecule of strychnine binds to the receptor and the 'additional' strychnine molecule in the dimer probably protrudes from the orthosteric binding sites of the receptor.},
  subject      = {Strychnin},
  language  = {en}
}
@phdthesis{Messerer2017,
  author    = {Messerer, Regina},
  title     = {Synthesis of Dualsteric Ligands for Muscarinic Acetylcholine Receptors and Cholinesterase Inhibitors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149007},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The study is dealing with the synthesis and pharmacological investigation of newly designed dualsteric ligands of muscarinic acetylcholine receptors belonging to the superfamily of G protein-coupled receptors. Such bipharmacophoric ligands combine the advantages of the orthosteric binding site (high-affinity) and of the topographically distinct allosteric binding site (subtype-selectivity) resulting in compounds with reduced side effects. This opens the way to a new therapeutic approach in the treatment of e.g. chronic pain, drug withdrawal, Parkinson`s and Alzheimer`s disease. Furthermore, the newly synthesized dualsteric compounds were pharmacologically investigated in order to get a better understanding of the activation and signaling processes in muscarinic acetylcholine receptors, especially with regard to partial agonism. The development of the "dynamic ligand binding" concept offers new perspectives for ligand binding and signaling at G protein-coupled receptors. GPCRs are no longer considered as simple on/off switches. Dualsteric ligands can bind in a dualsteric pose, reflecting an active receptor state as well as in a purely allosteric binding pose, characterized by an inactive receptor state resulting in partial agonism. The degree of partial agonism depends on the ratio of active versus inactive receptor populations. On this basis, orthosteric/orthosteric hybrid ligands consisting of the antagonist atropine and scopolamine, respectively, as well as of the agonist iperoxo and isoxazole, respectively, linked via different alkyl chain length were synthesized in order to investigate partial agonism (Figure 1). Figure 1: Structures of the synthesized iperoxo/isoxazole-atropine/scopolamine-hybrids. Furthermore, different sets of quaternary and tertiary homodimers consisting either of two iperoxo or two acetylcholine units were synthesized in order to study their extent on partial agonism (Figure 2). The two agonists were connected by varying alkyl chain length. Binding studies on CHO-hM2 cells of the quaternary compounds revealed that dimerization of the agonist results in a loss of potency. The iperoxo-dimers reached higher maximum effects on the Gi- as well as on the Gs pathway in comparison to the acetylcholine-dimers. Besides the choice of the orthosteric building block (potency of the agonist), the alkyl chain length is also crucial for the degree of partial agonism. Figure 2: Structures of the synthesized quat./tert. iperoxo/acetylcholine-homodimers. Quinolone-based hybrids connected to the superagonist iperoxo and to the endogenous ligand acetylcholine, respectively, linked through an alkyl chain of different length were synthesized in order to develop further partial agonists (Figure 3). FRET studies confirmed M1 subtype-selectivity as well as linker dependent receptor response. The greatest positive FRET signal was observed with quinolone-C6-iper resulting from a positive cooperativity between the two separated moieties, alloster and orthoster. However, the corresponding hybrids with a longer linker led to an inverse FRET signal indicating a different binding mode, e.g. purely allosteric, in contrast to the shorter linked hybrids. Furthermore, the flexible alkyl spacer was replaced by a rigidified linker resulting in the hybrid quinolone-rigid-iperoxo (Figure 3). FRET studies on the M1 receptor showed reduced FRET kinetics, resulting from interactions between the bulky linker and the aromatic lid, located between the orthosteric and allosteric binding site. A bitopic binding mode of the rigidified hybrid is presumed. For further clarity, mutational studies are necessary. Figure 3: M1-selective hybrid compounds. Another aim of this work was the design and synthesis of new hybrid compounds, acting as agonists at the M1 and M2 receptor and as inhibitors for AChE and BChE in the context of M. Alzheimer. Several sets of hybrid compounds consisting of different pharmacophoric units (catalytic active site: phthalimide, naphthalimide, tacrine; peripheric anionic site: iperoxo, isoxazole) linked through a polymethylene chain of varying length were synthesized. Tac-C10-iper (Figure 4), consisting of tacrine and the superagonist iperoxo linked by a C10 polymethylene spacer, was found to have excellent anticholinesterase activity for both AChE (pIC50 = 9.81) and BChE (pIC50 = 8.75). Docking experiments provided a structural model to rationalize the inhibitory power towards AChE. Additionally, the tacrine related hybrids showed affinity to the M1 and M2 receptor. Such compounds, addressing more than one molecular target are favorable for multifactorial diseases such as Alzheimer. Figure 4: Structure of the most active compound regarding anticholinesterase activity. In summary, the choice of the pharmacophoric units, their connecting point as well as the nature, length, and flexibility of the linker play an important role for the activity of designed bivalent ligands. A shorter linker length cannot bridge both binding sites simultaneously in contrast to longer linker chains. On the other hand, too long linker chains can result in unwanted steric interactions. Further investigations with respect to structural variations of hybrid compounds, with or without quaternary ammonium groups, are necessary in the light of drug development.},
  subject      = {Cholinesteraseinhibitor},
  language  = {en}
}
@phdthesis{HebronMwalwisi2018,
  author    = {Hebron Mwalwisi, Yonah},
  title     = {Assessment of Counterfeit and Substandard Antimalarial Medicines using High Performance Thin Layer Chromatography and High Performance Liquid Chromatography},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145821},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Although the prevalence of substandard and counterfeit pharmaceutical products is a global problem, it is more critical in resource-constrained countries. The national medicines regulatory authorities (MNRA) in these countries have limited resources to cater for regular quality surveillance programmes aimed at ensuring that medicines in circulation are of acceptable quality. Among the reasons explained to hinder the implementation of these strategies is that compendial monographs are too complicated and require expensive infrastructures in terms of environment, equipment and consumables. In this study it was therefore aimed at developing simple, precise, and robust HPLC and HPTLC methods utilizing inexpensive, readily available chemicals (methanol and simple buffers) that can determine the APIs, other API than declared one, and which are capable of impurity profiling. As an outcome of this study, three isocratic and robust HPLC and two HPTLC methods for sulfadoxine, sulfalene, pyrimethamine, primaquine, artesunate, as well as amodiaquine have been developed and validated. All HPLC methods are operated using an isocratic elution mode which means they can be implemented even with a single pump HPLC system and standard C18 columns. The densitometric sulfadoxine/sulfalene and pyrimethamine method utilizes standard TLC plates as well as inexpensive, readily available and safe chemicals (toluene, methanol, and ethyl acetate), while that for artesunate and amodiaquine requires HPTLC plates as well as triethylamine and acetonitrile due to challenges associated with the analysis of amodiaquine and poorly the detectable artesunate. These HPTLC methods can be implemented as alternative to those requiring HPLC equipment e.g. in countries that already have acquired densitometer equipment. It is understood that HPTLC methods are less sensitive, precise and accurate when compared to HPLC methods, but this hindrance can easily be addressed by sending representative samples to third party quality control laboratories where the analytical results are verified using compendial HPLC methods on a regular basis. It is therefore anticipated that the implementation of these methods will not only address the problem of limited resources required for medicines quality control but also increase the number of monitored targeted antimalarial products as well as the number of resource- constrained countries participating in quality monitoring campaigns. Moreover, the experiences and skills acquired within this work will be applied to other API groups, e. g. antibiotics, afterwards.},
  subject      = {Instrumentelle Analytik},
  language  = {en}
}
@phdthesis{Zilker2019,
  author    = {Zilker, Markus},
  title     = {The stability of finished pharmaceutical products and drug substances beyond their labeled expiry dates},
  doi       = {10.25972/OPUS-18069},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180695},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Upon approval of a drug, the stability of the API and the FPP has to be studied intensively because it determines the shelf-life. If a drug is found to be stable, the expiry date is arbitrary set to five years at the maximum, if a drug tends to undergo degradation, the expiry date is set shorter. The drug product must comply with predefined specifications in accordance with the ICH guidelines Q6A and Q6B during its entire market life. The content of the active substance is required to be within a specification of 95-105\% of its labeled claim until expiry corresponding to the ICH guideline Q1A(R2). However, there is little or scattered literature information addressing the stability of drug products beyond their expiry dates. The objective of this thesis was to study and assess the long-term stability of a collection involving numerous pure drug substances and ampoules manufactured in the 20th century. The content and the impurity profile were examined by means of appropriate analytical methods, mainly using liquid chromatography. The results were compared to data being available in the literature. Assessing the stability regarding the dosage form and the affiliation of the drug class was conducted. The experimental studies comprise the examination of 50 drug substances manufactured 20-30 years ago and 14 long expired ampoules which were older than 40 years in the time of analysis, exceeding many times the maximum shelf life of five years. For investigation of the solid drug substances, pharmacopoeial methods were applied as far as possible. Indeed, results of the study showed that 44 tested substances still complied with the specification of the Ph. Eur. with regard to the content and impurity profile, even after more than two decades of storage. For analysis of the injection solutions, HPLC-UV and HPLC-ESI/MS techniques were applied, commonly based on liquid chromatography methods of the Ph. Eur. for determination of related substances. Each method was further validated for its application to ensure accurate API quantification corresponding to ICH Q2(R1). Quite a few ampoules were identified to show surprisingly high stability. In spite of their age of 53-72 years, APIs such as caffeine, etilefrine, synephrine, metamizole sodium, furosemide, and sodium salicylate complied with the specified content that is valid nowadays, respectively. Nevertheless, typical degradation reaction, e.g. hydrolysis, oxidation, or isomerization, was observed in all remaining ampoules. Various degrees of hydrolysis were revealed for scopolamine, procaine, and adenosine triphosphate, the contents were decreased to 71\%, 70\%, and 15\% of the declared concentrations, respectively. In the epinephrine and dipyridamole ampoules, oxidative degradation has been occurred, finding respective API contents of more or less 70\%. For dihydroergotamine, excessive decomposition by epimerization was observed, resulting in an API content of 21\% and degradation by isomerization was found in lobeline, still containing 64\% of the labeled claim. In conclusion, supported by the data of the present studies and the literature, defining and authorizing a longer shelf-life may be applicable to numerous pharmaceuticals which should be considered by pharmaceutical manufacturers and regulatory authorities, if justified based on stability studies. A general extension of the shelf-lives of drug products and the abolishment or extension of the maximum shelf-life limit of five years would prevent disposing of still potent medications and save a lot of money to the entire health care system.},
  subject      = {Stabilit{\"a}t},
  language  = {en}
}
@phdthesis{Skaf2018,
  author    = {Skaf, Joseph},
  title     = {Antileishmanial and antitrypanosomal compounds from \(Achillea\) \(fragrantissima\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167841},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {This PhD thesis is dealing with the bioassay-guided fractionation of a dichloromethane extract of the aerial parts of Achillea fragrantissima with the aim of isolation and structure isolation of the antileishmanial and/or antitrypanosomal principles in the plant.},
  subject      = {Schafgarbe <Gattung>},
  language  = {en}
}
@phdthesis{Kuehnreich2016,
  author    = {K{\"u}hnreich, Raphael},
  title     = {Development and Validation of Methods for Impurity Profiling of Amino Acids},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145718},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The requirements for the impurity profiling of substances for pharmaceutical use have become greater over time. They can be accomplished by the use of modern instrumental analysis techniques, which have been evolved in the last decades. New types of columns with HILIC, mixed-mode and chiral stationary phases are suitable for the separation of all kinds of substances mixtures, that were previously hardly possible with the use of common reversed phase columns. Modern, almost universal detectors like CAD, ELSD and CNLSD can be applied for a sensitive detection of substances without a chromophore. However, in addition to some small individual disadvantages to these methods, the costs are high and applications are still kind of rare. Thus, the introduction of these devices at a broader level has not yet taken place. While this presumably will change over time, there is a need for methods that enable the impurity profiling of challenging substances with widespread analytics devices. Methionine is a substance with hydrophobic and hydrophilic impurities. With the help of a mixed-mode stationary phase, which is a combination of a reversed phase and a strong cationic exchanger, the separation of all putative impurities was found possible with good sensitivity and selectivity. The method requires apart from the column only standard isocratic HPLC equipment and was successfully validated. The evaluation of the enantiomeric purity of amino acids is challenging. Two approaches were made. The first method utilizes CE by means of in-capillary derivation with OPA and the subsequent separation with a cyclodextrin. With the use of OPA/NAC and γ-cyclodextrin, a simple and cost-effective method for the indirect enantioseparation of 16 amino acids was developed. With the second approach, racemic amino acids can be analyzed with HPLC and in-needle derivatization. For this, different columns and chiral thiols were evaluated and the chromatographic parameters were optimized. A method with OPA/NIBLC, a pentafluorophenyl column made the enantioseparation of 17 amino acids feasible. A LOQ of the minor enantiomer down to 0.04 \% can be achieved with UV spectrophotometric detection. A similar method was developed for impurity profiling of L-amino acids. This can be used alternatively for the amino acid analysis performed by the European Pharmacopoeia. A simple, robust, precise and accurate method for the evaluation of impurities in glyceryl trinitrate solution was developed and validated. The four impurities of glyceryl trinitrate are separated by means of an acetonitrile-water gradient and the assay for this substance is also possible.},
  subject      = {Aminos{\"a}uren},
  language  = {en}
}
@phdthesis{Slopianka2020,
  author    = {Slopianka, Markus},
  title     = {Evaluation von Gallens{\"a}uren als Biomarker f{\"u}r Lebertoxizit{\"a}t in der pr{\"a}klinischen Arzneimittelentwicklung},
  doi       = {10.25972/OPUS-20462},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204627},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Die Detektion Arzneimittel-induzierter Lebersch{\"a}digung (engl. DILI - Drug induced liver injury) stellt eine Herausforderung in der pr{\"a}klinischen Entwicklung von Arzneistoffen dar. Die zur Verf{\"u}gung stehenden konventionellen klinisch-chemischen Marker, wie Alanin-Aminotransferase (ALAT), Aspartat-Aminotransferase (ASAT) und Alkalische Phosphatase (APh), zeigen z. B. bei minimaler bis leichter Leberpathologie keine Ver{\"a}nderungen im Serum an und besitzen somit nur eine geringe Sensitivit{\"a}t f{\"u}r den fr{\"u}hzeitigen Nachweis einer Lebertoxizit{\"a}t. Des Weiteren besitzen klinisch-chemische Serummarker gleichzeitig eine geringe Spezifit{\"a}t und sind somit f{\"u}r die Differenzierung unterschiedlicher Lebertoxizit{\"a}ten nur limitiert geeignet. Neben den beschriebenen diagnostischen Herausforderungen k{\"o}nnen u. a. auch histopathologische Befunde in der Leber, ohne eine Ver{\"a}nderung der klinisch-chemischen Serummarker auftreten und umgekehrt. Die Histopathologie ist als Goldstandard zwar spezifisch, als invasive Technik f{\"u}r eine Verlaufskontrolle in toxikologischen und klinischen Studien aber ungeeignet. In den vergangenen Jahren lieferten Studien zum Gallens{\"a}ure-Profiling mittels Fl{\"u}ssigkeitschromatographie-Tandem-Massenspektrometrie (LC-MS/MS) mit Modellsubstanzen, die unterschiedliche Formen einer Lebertoxizit{\"a}t in Ratten induzierten Hinweise, dass individuelle Gallens{\"a}uren ein diagnostisches Potential f{\"u}r die Bewertung einer Lebersch{\"a}digung besitzen. Ziel dieser Arbeit ist es, dass Gallens{\"a}ure-Profiling in die vorgeschriebene Diagnostik der Lebertoxizit{\"a}t in der pr{\"a}klinischen Arzneimittelentwicklung zu implementieren und zu bewerten, ob diese Marker einen wertvollen Beitrag zur Charakterisierung einer Lebertoxizit{\"a}t leisten k{\"o}nnen. Hierzu wurde eine quantitative LC-MS/MS-Methode etabliert und validiert, die es erm{\"o}glicht, 20 verschiedene endogene Gallens{\"a}uren in Ratten zu analysieren. Die quantitative Analytik erm{\"o}glichte eine selektive Bestimmung von prim{\"a}ren, konjugierten und sekund{\"a}ren Gallens{\"a}uren. F{\"u}r die Quantifizierung der individuellen Gallens{\"a}uren wurden 2 MRM-{\"U}berg{\"a}nge bestimmt. Zur Bestimmung des Arbeitsbereiches wurden 20 Referenzstandards von Gallens{\"a}uren verwendet. Eine Kalibrierung mit sieben Kalibrierpunkten in aufsteigender Konzentration wurde f{\"u}r die Bestimmung der endogenen Konzentrationen genutzt. Zur Kompensation des Matrixeffektes wurden 10 isotopenmarkierte interne Standards in die Analytik eingef{\"u}gt. Die Reproduzierbarkeit laufender Messungen wurde durch eingef{\"u}gte Qualit{\"a}tskontrollen (QCs) in drei verschiedenen Konzentrationsbereichen {\"u}berwacht. Es wurde ein Gallens{\"a}ure-Profiling mittels LC-MS/MS im Plasma und Lebergewebe von Ratten, die mit verschiedenen Arzneimitteln behandelt wurden, durchgef{\"u}hrt. Histopathologische Zusammenfassung Untersuchungen konnten aufzeigen, dass sich in den Lebern von m{\"a}nnlichen Ratten, die mit dem Arzneimittel Amitriptylin {\"u}ber 14 Tage behandelt wurden, eine makrovesikul{\"a}re Steatose in der Leber manifestierte. Die klassischen Serummarker, wie ALAT, ASAT und Gamma-Glutamyltransferase (γGT), konnten diese Art des Leberschadens nicht detektieren. Dagegen erh{\"o}hten sich die Konzentrationen Glycin-konjugierter Gallens{\"a}uren mit parallel absinkenden Konzentrationen von Taurin-konjugierten Gallens{\"a}uren im Lebergewebe behandelter Ratten. Gleichzeitig ergaben sich signifikant erh{\"o}hte Konzentrationen der prim{\"a}ren Gallens{\"a}uren CA und CDCA im Plasma behandelter Ratten. Andere Gallens{\"a}ure-Profile konnten nach einer Methapyrilen-induzierten Leberzellnekrose mit hepatobili{\"a}rer Sch{\"a}digung beobachtet werden. Nach einer 14-t{\"a}gigen Behandlungsphase mit 80 mg/kg KG Methapyrilen, erh{\"o}hten sich die Konzentrationen von 11 Gallens{\"a}uren im Lebergewebe behandelter Tiere. Gleichzeitig stiegen die Konzentrationen von allen 20 individuellen Gallens{\"a}uren im Plasma behandelter Ratten an. Zus{\"a}tzlich zur quantitativen Analyse von Gallens{\"a}uren mittels LC-MS/MS wurde die Expression von Genen der Gallens{\"a}ure-Biosynthese, des Gallens{\"a}ure-Transports und die Regulation der Gallens{\"a}ure-Hom{\"o}ostase mittels Multiplex-Analyse untersucht. Die erh{\"o}hte Expression von Genen f{\"u}r Efflux-Transporter der Multidrug Resistance-Related Protein (MRP)-Familie deutet auf einen gesteigerten Abtransport von Gallens{\"a}uren ins Blut hin und korrespondierte mit erh{\"o}hten Gallens{\"a}ure-Konzentrationen im Plasma der behandelten Ratten. Des Weiteren wurden die Erkenntnisse der Gallens{\"a}ure-Profile aus den tierexperimentellen Studien als Grundlage genutzt, um Arzneimittel-induzierte Lebertoxizit{\"a}t auf ein zellbiologisches In-vitro-System zu {\"u}bertragen. Es wurden In-vitro-Experimente mit prim{\"a}ren Rattenhepatozyten zwischen zwei Kollagenmatrices (Sandwich-Kultivierung) durchgef{\"u}hrt. Dieses etablierte System wird u. a. f{\"u}r Untersuchungen an hepatobili{\"a}ren Transportsystemen (z. B. Bile Salt Export Pump, BSEP) genutzt. Das Gallens{\"a}ure-Profiling in den Zellkultur{\"u}berst{\"a}nden belegt, dass die prim{\"a}ren Hepatozyten konjugierte Gallens{\"a}uren bilden, dass sie bei einer Inkubation mit prim{\"a}ren Gallens{\"a}uren diese verstoffwechseln und dadurch, neben den bereits vorhandenen Gallens{\"a}uren, weitere konjugierte Gallens{\"a}uren produzieren. Eine Exposition mit den Hepatotoxinen Troglitazon und Methapyrilen f{\"u}hrte zu Ver{\"a}nderungen in der Gallens{\"a}ure-Hom{\"o}ostase der Hepatozyten. In den In-vivo-Experimenten wurde eine Methapyrilen-induzierte Nekrose mit hepatobili{\"a}rer Sch{\"a}digung in den behandelten Ratten festgestellt. Bei der Behandlung mit Methapyrilen ergaben sich starke Konzentrationsanstiege der Gallens{\"a}uren im Plasma (u. a. von GCA und TCA), die mit den histopathologischen Befunden korrelierten. Anhand dieser Daten und der Zusammenfassung pharmakokinetischen Eigenschaften von Methapyrilen wurde ein Studiendesign f{\"u}r Rattenhepatozyten in Sandwich-Kulturen entwickelt, um eine initiale Absch{\"a}tzung der Konzentrationsver{\"a}nderungen von Gallens{\"a}uren im In-vitro-Testsystem durchzuf{\"u}hren. Ab Tag 8 der Behandlung kam es zu einem erh{\"o}hten Anstieg der GCA- und TCA-Konzentrationen im Zellkulturmedium. Daher besitzt das In-vitro-Testsystem m{\"o}glicherweise das Potential, tierexperimentelle Studien bei der Bewertung einer Hepatotoxizit{\"a}t zu unterst{\"u}tzen oder sogar zu reduzieren. Insgesamt zeigen diese Ergebnisse aus dieser Arbeit, dass Gallens{\"a}ure-Profiling in m{\"a}nnlichen und weiblichen Ratten eine geeignete Methode zur Detektion und Differenzierung von Lebersch{\"a}den ist. Die Technologie ist flexibel einsetzbar und kann bereits etablierte Testverfahren, wie die Bestimmung von Serummarkern in der Klinischen Chemie und die Histopathologie unterst{\"u}tzen. Damit besitzt das Gallens{\"a}ure-Profiling das Potential, die Bewertung beim Nachweis und bei der Charakterisierung einer Lebertoxizit{\"a}t im Rahmen der Evaluierung von pr{\"a}klinischen Arzneimittelkandidaten zu verbessern.},
  subject      = {Hepatotoxizit{\"a}t},
  language  = {de}
}
@phdthesis{Theiss2019,
  author    = {Theiss, Christiane},
  title     = {Qualitative Charakterisierung polydisperser Macrogole sowie strukturell verwandter Hilfsstoffe mittels HPLC-CAD},
  doi       = {10.25972/OPUS-17927},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179274},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The class of macrogols and macrogol-based excipients, i.e. macrogol fatty alcohol ethers, macrogol fatty acid esters, and polysorbates, plays an important role in modern galenic formulations. Formerly used as simple emulsifiers, they are nowadays utilized in fields such as targeted drug release to increase bioavailability, and as solubilizers for complex systems. For these multifaceted applications, and regarding the polydisperse structures of the macrogols, a reproducible and significant analytical procedure is required. For the characterization of excipients, the European Pharmacopoeia (Ph. Eur.) provides some compendial protocols which are able to describe the number of functional groups present in the substance. Some examples of these bulk parameters are the hydroxyl value, the iodine value, the peroxide value, or the acid value. Thus, these bulk parameters allow an overview of the average molar weight or possible degradation processes (e.g. autoxidation), but they provide no further information about the polymeric distribution which can heavily depend on the manufacturing process. Furthermore, bulk parameter investigations are very time-consuming and prone to errors due to their stringent reaction processes and numerous reaction steps. Since several years, the HPLC has been the gold standard of pharmaceutical analytics particularly due to the fact of automation. Coupled to UV detection, it offers the opportunity for a quick, easy, and robust analysis for many drugs. In the field of excipients, the development progress of HPLC-analysis is much slower due to the fact that most excipients lack a UV-chromophore. The application of the highly sensitive mass spectrometry would be eligible for detection but is rather complex and expensive. However, the development of the aerosol-based detectors such as the ELSD (evaporative light scattering detection), the CAD (charged aerosol detection), and the NQADTM (nano quantity aerosol detection) enables the application of HPLC for analyzing non-chromophoric substances. This work aimed to develop a generic HPLC-CAD method to analyze a wide range of macrogols and macrogol-based excipients. The separation was performed on a C18-column. A gradient method was developed based upon several linear gradient steps in order to be able to separate the different chain lengths. The mobile phases were water and acetonitrile, respectively, to which 0.1\% formic acid was added. Macrogols in the average size range of PEG 300 to PEG 3000 were separated with acceptable resolution. The separation results were verified by mass spectrometry for PEG 300 - 1500. Five saturated and two non-saturated fatty acids, as well as two fatty alcohols of different chain lengths were successfully separated. 13 macrogol-based excipients were analyzed with the developed method and separated successfully. The macrogol fatty alcohol ethers, macrogol stearates, and polysorbates were separated to sufficient extent to analyze the polymeric distribution. The free PEGs in the excipients were separated and identified. Based on these free PEGs, different manufactural processes could be determined. Depending on the average chain lengths of the processed PEGs, the free fatty acids or alcohols could be identified and separated from the esters or ethers, respectively. For the smaller average chain lengths, the free fatty acids and alcohols coeluted with the esters and ethers. Macrogol glycerol hydroxy stearate (Cremophor® RH40) was separated into its components except for the linear monoesters which partially coeluted with the free PEGs, and the glycerol triesters which showed effects of size exclusion. The developed method was also used for stability tests of the non-saturated fatty acids, i.e. oleic and linoleic acid. Here, the fatty acid solutions were chemically (hydrogen peroxide) and thermally (60 °C) stressed and analyzed after different time spans. A time and temperature dependent degradation was observed. An assignment of some degradation products was performed by determining the m/z values with mass spectrometry. The method proved to be capable of separating the degradation products of the main substance and allows to estimate the dimension of degradational processes and partly identify the structures of the degradational products. In general, the provided method offers a good basis for analyzing and characterizing a wide field of substance classes. It provides an extension of bulk parameters (e.g. hydroxyl value) with a reduction of analytical effort. It offers a good starting point for more specific observations such as long-term stability or other related substance classes.},
  subject      = {HPLC},
  language  = {de}
}
@phdthesis{Munz2019,
  author    = {Munz, Martin Reinhold},
  title     = {Individualization of drug therapy considering pharmacokinetic and clinical factors},
  doi       = {10.25972/OPUS-17339},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173396},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In the „Position Paper of the Division of Clinical Pharmacy of the German Pharmaceutical Society (DPhG)" clinical pharmacy is defined as the science and practice of the rational use of drugs1, which includes the individualization of drug therapy. Clinical pharmacists therefore need a profound knowledge of the pharmacokinetic properties of relevant drugs, and clinical factors that are influencing these properties. Against the background of individualizing drug therapy, pharmacokinetic and clinical factors are studied in this thesis. In order to obtain an overview of the existing data on the pharmacokinetics of imipenem / cilastatin and meropenem in critically ill patients, a literature review for each of these carbapenem antibiotics was performed. These reviews included studies in critically ill patients as well as studies in healthy volunteers. While the reported results of studies in healthy volunteers had a small variability, studies in critically ill patients show significant differences in the resulting pharmacokinetics. These differences were not only between, but also within these studies, resulting in a high variability of the pharmacokinetic parameters of the carbapenems in critically ill patients. Furthermore, the results of studies in critically ill patients indicate that clinical factors and in particular renal function have different effects on the pharmacokinetics of imipenem and cilastatin. A therapeutic drug monitoring (TDM) program for antibiotics was initiated in an intensive care unit. The calculation of the pharmacokinetics of imipenem / cilastatin and meropenem was carried out with a population pharmacokinetic approach (POP-PK) and in addition with a non-compartmental approach (NCA). The POP-PK analysis showed that the pharmacokinetics of imipenem and cilastatin could be described adequately with a 1-compartment model. The resulting mean total body clearance (CL) of imipenem and cilastatin was 11.6 L/h (4.24 to 27.5) and 6.14 L/h (0.520 to 26.6 L/h). The nonrenal clearance was estimated to be 5.30 L / h (24.9\% CV) for imipenem and 0.138 L / h (33.3\% CV) for cilastatin. The results of the NCA were in good agreement with the results of the POP-PK approach, as the NCA resulted in an imipenem clearance of 15.5 ± 7.3 L / hr and cilastatin clearance of 10.1 ± 9.9 L / h. The individual clearances resulting from the different pharmacokinetic approaches were in good correlation showing correlation coefficients (r) of 0.882 (p <0.001) and 0.908 (p <0.001) for imipenem and cilastatin. In summary, this study identified and quantified significant differences between the individual clearance mechanisms of imipenem and cilastatin. This is particularly true for patients with impaired renal function and sepsis. As imipenem / cilastatin is only available in a fixed dose combination, those patients might be treated inadequately with this combination. The great variability in the pharmacokinetics of imipenem and cilastatin in septic patients underscores the importance of a TDM program of both substances. For meropenem, a PK/PD model was developed that predicts the concentration gradients of meropenem, serum creatinine, C-reactive protein and procalcitonin simultaneously. A non-linear relationship between the clearance of creatinine and meropenem was identified and the resulting equation for the calculation of the total body clearance of meropenem (for a 70 kg patient) was: 0.480 L/h + 9.86 L/h. (CLCR/6L/h)0.593, with 0.480 L/h representing the nonrenal clearance of meropenem. The resulting mean meropenem clearance of the NCA was 11.9 ± 8.7 L/h. The individual clearances resulting from the different pharmacokinetic approaches were poorly correlated showing a correlation coefficient (r) of 0.502 (p <0.001). In summary, this study showed a non-linear relationship of meropenem clearance and creatinine clearance. The model shows that the renal function may change rapidly and to a significant extent in patients with sepsis and septic shock, which in turn, underscores that creatinine concentrations are not in steady state in these patients. Conversely, dose adjustment based on creatinine values might lead to inappropriate therapy. This underlines the importance of a TDM program for meropenem in critically ill patients. The two most important considerations when choosing an antibiotic for the prophylaxis of postoperative bone infections are its activity against the whole spectrum of bacteria, which might be involved in bone infections, and its ability to penetrate bone tissue and thus to achieve concentrations above the minimum inhibitory concentration (MIC) of the corresponding pathogens. In order to gain information on this data, a study was conducted which investigated the pharmacokinetics of ampicillin / sulbactam in plasma, cortical and cancellous bone. Pharmacokinetic parameters in plasma were determined using NCA. The bone penetration represents the ratio of the concentration in the bone tissue to plasma concentration at the time of bone removal. The resulting half-life of ampicillin and sulbactam in plasma was 1.60  0.37 h and 1.70  0.42 h. The elimination of both substances was in a good correlation with creatinine clearance and resulted in correlation coefficients (r) of 0.729 (p = 0.003) for ampicillin and 0.699 (p = 0.005) for sulbactam. The mean clearance and the mean volume of distribution of ampicillin and sulbactam were 10.7  3.9 and 10.3  3.3 L/h, and 23.9  7.9 and 24.3  6.8 L. The mean concentrations of ampicillin in the cortical and cancellous bone were 6.60  4.22 and 10.15  7.40 µg/g, resulting in bone penetration ratios of 9.1  5.7 and 16.2  16.9 \%. For sulbactam the corresponding concentrations were 3.91  2.52 and 5.73  4.20 µg/g, resulting in bone penetration ratios of 10.6  6.3 and 17.5  16.1 \%. In summary, this study shows that the bone penetration of both substances is on average rather unsatisfactory and has a high variability, which can lead to inadequate bone concentrations for the prophylaxis of bone infections. One factor that could be identified for the penetration of both substances into cancellous bone was the period between the application of the drug and the removal of the bone. Therefore, a time interval between the administration of the antibiotic and the incision should be considered. Immunosuppression is a risk factor for the development of various malignancies, including hematologic diseases. While the relationship between the use of immunosuppressive therapy with methotrexate and the development of an Epstein-Barr virus (EBV) associated lymphoproliferative disease (LPD) has been well established, this connection is less evident for immunosuppressive therapy with azathioprine. The patient presented by us was immunosuppressed with azathioprine for autoimmune hepatitis. The development of an EBV-associated Hodgkin-like lymphoma under this immunosuppressive therapy and especially the regression of the lymphoma after cessation of azathioprine confirms the relationship between this immunosuppressant, EBV-infection and the development of Hodgkin-like lymphoma. Therefore, albeit in rare cases, azathioprine-related lymphomas may respond to mere cessation of immunosuppressive therapy without need for chemotherapy. Apart from viral infections, drugs are a major cause of acute liver failure. Due to the lack of specific symptoms or tests, it is difficult to diagnose a drug-induced liver injury. We report a case of a young patient in whom different antibiotics, the analgesic and antipyretic acetaminophen or a combination of these drugs may have led to DILI resulting in life-threatening ALF. Based on this case report, we describe a procedure to exclude non-drug related causes and discuss the hepatotoxic potential of the involved drugs in this case.},
  subject      = {Pharmakokinetik},
  language  = {en}
}
@phdthesis{Bank2014,
  author    = {Bank, Stephanie},
  title     = {LC-ESI und MALDI-Massenspektrometrische Analyse nativer und derivatisierter Zucker und Glykane},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-106085},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Glykane sind weitverbreitete Biomolek{\"u}le, die meist in Form von Glykokonjugaten, wie beispielsweise als Glykoproteine oder Glykolipide, vorliegen. Durch die Interaktion von Glykanen mit Glykan-bindenden Proteinen wird eine Vielzahl an biochemischen Prozessen ausgel{\"o}st, sowohl physiologischer, als auch pathologischer Art. Die Aufkl{\"a}rung der beteiligten Glykanstrukturen ist daher nicht nur wichtig f{\"u}r das Verst{\"a}ndnis dieser Prozesse, sondern kann auch Hinweise auf verschiedene Erkrankungen geben. Die Identifizierung von Glykanstrukturen kann {\"u}ber verschiedene Wege erfolgen. In der instrumentellen Analytik spielt dabei vor allem die ESI- und MALDI Massenspektrometrie eine wichtige Rolle, da diese sowohl f{\"u}r Detektion, als auch Fragmentierung großer Biomolek{\"u}le geeignet sind. Um die Analyse von Zuckern mittels chromatographischer und massenspektrometrischer Methoden zu erleichtern, werden h{\"a}ufig Derivatisierungsreagenzien eingesetzt. Diese verringern die Polarit{\"a}t der Zucker und erleichtern die Detektion durch das Einbringen von Chromo- oder Fluorophoren. Zur Derivatisierung am reduzierenden Terminus von Glykanen und Zuckern eignen sich vor allem Aminierungsreagenzien oder Hydrazide. Hydrazide haben gegen{\"u}ber anderen Derivatisierungsreagenzien den Vorteil einer einfachen, salzfreien Umsetzung, aus der ein stabiles Derivat mit geschlossenem terminalen Zuckerring hervorgeht. F{\"u}r die vorliegende Arbeit wurde die Derivatisierung mit den neuen Hydrazid Reagenzien INH und BINH, sowie dem bereits von Dr. P. Kapkov{\´a} bearbeiteten BACH untersucht. Als Vergleich dienten die underivatisierten Kohlenhydrate, wie auch das standardm{\"a}ßig eingesetzte Aminierungsreagenz 2-AB. Dabei sollte das Ver-halten verschiedener Zucker und Glykane in Bezug auf chromatographische Trennung, Signalintensit{\"a}t und Fragmentierung analysiert werden. Zun{\"a}chst wurde die Umsetzung von Mono-, Di- und Trisacchariden mit den neuen Derivatisierungsreagenzien INH und BINH optimiert. Dadurch konnte bei beiden Substanzen die komplette Umsetzung der Zucker in ihre Derivate gew{\"a}hrleistet werden. Auch die Derivatisierung mit Hilfe der Mikrowelle konnte bei INH erfolgreich durchgef{\"u}hrt werden. Auf diese Weise ließ sich die Reaktionszeit, im Vergleich zu den im Thermo-mixer® ben{\"o}tigten 90 Minuten, auf 20 Minuten verk{\"u}rzen. Aufgrund der großen Men-gen an Zucker und Derivatisierungsreagenz, die f{\"u}r die Umsetzung in der Mikrowelle n{\"o}tig sind, war der Versuch jedoch nur f{\"u}r INH geeignet. Im n{\"a}chsten Schritt wurde das Trennverhalten der verschiedenen Mono-, Di- und Tri-saccharid-Derivate auf RP-C18- und HILIC-Phasen untersucht. Bei den Monosaccha-riden konnte durch keines der Derivate eine vollst{\"a}ndige Trennung auf einer der Pha-sen erreicht werden. Das beste Ergebnis wurde durch INH auf der HILIC-S{\"a}ule erzielt, doch auch dort konnten die Epimere Glucose, Mannose und Galactose nicht vollst{\"a}n-dig separiert werden. Die Trennung der Disaccharide Maltose, Cellobiose und Lactose konnte auf der HILIC-Phase mit allen Derivaten außer BACH erfolgreich durchgef{\"u}hrt werden, auf der RP-C18 erwies sich dagegen nur 2-AB als geeignet. Bei den Trisac-chariden 3'SLN und 6'SLN konnten sowohl underivatisierte Zucker, als auch s{\"a}mtliche Derivate mittels HILIC getrennt werden. Auch auf der C18-Phase war eine Trennung der BINH, BACH und 2-AB-Derivate m{\"o}glich. Des Weiteren konnte durch die Derivati-sierungen die Signalintensit{\"a}t gegen{\"u}ber den underivatisierten Zuckern deutlich gesteigert werden. Nach ihrer Trennung lassen sich massegleiche Di- und Trisaccharide anhand des Fragmentierungsmusters unterscheiden. W{\"a}hrend bei den underivatisierten Disaccha-riden Maltose, Cellobiose und Lactose die charakteristischen Fragmente nur schwach sichtbar waren, konnte mit Hilfe der Hydrazide INH, BINH und BACH die Differenzie-rung deutlich erleichtert werden. Die 2-AB-Derivatisierung zeigte dagegen keine Ver-besserung der Fragmentierungseigenschaften. Bei der Unterscheidung der Trisaccharide 3'SLN und 6'SLN waren ebenfalls sowohl underivatisierte, als auch Hydrazid-derivatisierte Zucker im Vorteil gegen{\"u}ber den 2-AB-Derivaten. Die Derivatisierung der N-Glykane von Ribonuclease B und Ovalbumin f{\"u}hrte bei der Analyse mittels MALDI-TOF zu einer deutlichen Steigerung der Sensitivit{\"a}t. Beispiels-weise ließen sich bei den Glykanen des Ovalbumins durch die Derivatisierungen drei zus{\"a}tzliche Strukturen im Vergleich zu den nativen Glykanen detektieren. Auch das Fragmentierungsverhalten der Glykane am MALDI-TOF/TOF konnte mit Hilfe der Derivatisierungen erheblich verbessert werden. Besonders die Umsetzung mit BINH f{\"u}hrte zu einer Vielzahl charakteristischer Ringfragmente, wodurch die Aufkl{\"a}rung der verschiedenen Glykanstrukturen deutlich vereinfacht wurde. Auch im Vergleich zu 2 AB zeigten die Hydrazid-Derivate sowohl bessere Fragmentierungseigenschaften, als auch eine einfachere Handhabung f{\"u}r die Messung mittels MALDI-MS. Eine weitere M{\"o}glichkeit zur Identifikation von Glykanstrukturen liegt in der spezifischen Bindung durch Lektine. Diese Untersuchung gibt des Weiteren auch einen Hinweis auf funktionelle Eigenschaften der Glykane. Daf{\"u}r wird die hohe Affinit{\"a}t von Biotin-haltigen Derivatisierungsreagenzien zu Avidin und Streptavidin genutzt. Nach der auf diese Weise erfolgten Immobilisierung der Glykane k{\"o}nnen diese mittels spezifischer Lektine nachgewiesen werden. Die Eignung des neuen Derivatisierungsreagen-zes BINH f{\"u}r diese Zwecke wurde anhand eines Glykan-Arrays getestet. Dadurch ließ sich best{\"a}tigen, dass BINH-derivatisierte Glykane und Zucker sowohl in der Lage sind an Streptavidin zu binden, als auch durch Lektine nachgewiesen werden k{\"o}nnen. Daher kann davon ausgegangen werden, dass BINH grunds{\"a}tzlich f{\"u}r den Einsatz in bio-chemischen Methoden geeignet ist. Zusammenfassend l{\"a}sst sich sagen, dass die Derivatisierung von Kohlenhydraten mit INH, BINH und BACH zu einer deutlichen Verbesserung der Trenn- und Fragmentierungseigenschaften f{\"u}hrten. Dadurch konnten Identifizierung und Strukturanalyse sowohl von kleinen Zuckern, als auch von Glykanen erleichtert werden. Im Vergleich zu dem Standard-Derivatisierungsreagenz 2-AB zeigten die Hydrazide nicht nur im Bereich der Fragmentierungen, sondern auch durch die einfachere Derivatisierungsreaktion wesentliche Vorteile.},
  subject      = {MALDI-MS},
  language  = {de}
}
@phdthesis{Ilko2015,
  author    = {Ilko, David},
  title     = {The use of charged aerosol detection for the analysis of excipients and active pharmaceutical ingredients},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118377},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The Corona® charged aerosol detector (CAD) is an aerosol-based detector first de-scribed by Dixon and Peterson in 2002. It is capable of detecting compounds inde-pendent from their physico-chemical properties presumed the analyte is sufficiently non-volatile. Consequently, the CAD is often applied to the analysis of substances that do not possess a suitable UV chromophore. Major drawbacks are however, the detector signal is non-linear and depending on the content of organic solvent in the mobile phase. This thesis tried to explore possible applications of the CAD for pharmaceutical analysis. Therefore, several substances from different compound classes were in-vestigated. Newly developed or existing methods were validated. Thus the perfor-mance of the CAD could be examined. Both assay and impurity determination were evaluated for their compliance with ICH Q2(R1) "Validation of Analytical Proce-dures" and the "Technical Guide for the Elaboration of Monographs". In the course of the establishment of reference substances at the EDQM, a generic screening method for the identification of organic and inorganic pharmaceutical counterions was needed. An HPLC-CAD method developed by Zhang et al. was therefore investigated for its suitability for pharmacopoeial purpose. Method valida-tion was performed. It was found that 23 ions could be separated and detected. Iden-tification was achieved via retention time of an authentic standard of the corre-sponding ions. Alternatively, peak assignment was performed by determination of the exact mass using TOF-MS. Ions could be quantified as impurities or for stoichi-ometric purpose. For the impurity control in topiramate, the performance characterstics of the CAD were compared to that of an ELSD. CAD was superior to ELSD in terms of repeata-bility, sensitivity and linearity. However, impurities could be quantified with satisfac-tory accuracy with both detectors. The application of the ELSD was not feasible due to non-reproducible spike peaks eluting after the principle peak in the chromatogram of the test solution. One of the impurities, topiramate impurity A (diacetonide), gave no or a vastly diminished signal in the ELSD and the CAD, respectively. It is evapo-rated during the detection process due to its relatively high vapor pressure. The re-sponse could be enhanced by a factor of nine via post-column addition of acetoni-trile and a lower nebulizer temperature. As the response of topiramate impurity A was still about thousand-fold lower than the response of all other impurities, its quantification was not feasible. Additionally, the HPLC-CAD was successfully vali-dated as an assay procedure for topiramate. There seems to be a great potential in the application of the CAD to the analysis of excipients as most compounds do not possess a suitable UV chromophore. Here, a simple and rapid HPLC-CAD method for the determination of polidocanol (PD) was developed. The method was successfully validated as a potential assay procedure for the Ph. Eur. as none is described in either of the two PD monographs. The same method was applied to the determination of the PD release from a pharmaceutical polymer matrix. A method for the determination of the fatty acid (FA) composition of polysorbate 80 (PS80) was developed and validated. Using the CAD and mass spectrometry, we were able to identify two new FAs in 16 batches from four manufacturers. All batch-es complied with pharmacopoeial specification. Furthermore, the overall composi-tion of the different PS80 species ("fingerprinting") and the peroxide content were determined. In addition to the chemical characterization, functionality related charac-teristics (FRCs) were determined. Correlations between chemical composition and FRCs were found. The validation data of the above mentioned methods suggests that the CAD repre-sents a viable detection technique for pharmaceutical analysis. The CAD was suffi-ciently sensitive for non-volatile analytes. Impurity control down to concentrations of 0.05 or 0.03\%, as demanded by ICH Q3A (R2), is achievable. However, the response of semi-volatile compounds may be drastically diminished. It could be confirmed that the response of the CAD is linear when the range does not exceed two orders of magnitude. Exceptions may be observed depending on the actual method setup. When the measuring range is sufficiently narrow, quantification can be done using single-point calibration which is common practice in pharmaceutical anlysis. Impuri-ties may also be quantified against a single calibration solution. However, correction factors may be needed and the accuracy is considerably lower compared to an as-say method. If a compound is to be quantified over a large concentration range, log-log transformation of the calibration curve is needed and a decreased accuracy has to be accepted.},
  subject      = {Analyse},
  language  = {en}
}
@phdthesis{Wiest2015,
  author    = {Wiest, Johannes},
  title     = {Synthese und Charakterisierung neuer Ionischer Fl{\"u}ssigkeiten zur Verbesserung der Aufl{\"o}sungsrate und L{\"o}slichkeit eines schwer wasserl{\"o}slichen Wirkstoffes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121733},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Ionische Fl{\"u}ssigkeiten (engl. Ionic Liquids = IL) sind organische Salze mit einem Schmelzpunkt von unter 100 °C und bieten einen interessanten Ansatz um die orale Bioverf{\"u}gbarkeit von schlecht wasserl{\"o}slichen Arzneistoffen zu verbessern. Aufgrund seiner schlechten Wasserl{\"o}slichkeit wurde aus dem Wirkstoff BGG492 der Novartis AG eine Ionische Fl{\"u}ssigkeit (IL) mit dem sterisch anspruchsvollen Gegenion Tetrabutylphosphonium hergestellt. Die IL ist ein amorpher, glasartiger Feststoff mit einem Schmelzpunkt von 57 °C. Die freie S{\"a}ure (FS), das Kaliumsalz (BGG-K+) und die IL (siehe Abb. 69) wurden in festem Zustand mittels polarisationsmikroskopischen Aufnahmen, R{\"o}ntgen-Pulverdiffraktometrie, R{\"o}ntgenkristallstrukturanalysen, Infrarot-Spektroskopie und Festk{\"o}rper-NMR-Spektroskopie untersucht. Der ionische Charakter der IL in festem Zustand konnte mittels Bandenverschiebung der deprotonierten Sulfonamidgruppe im IR-Spektrum best{\"a}tigt werden. In der R{\"o}ntgenkristallstrukturanalyse konnte gezeigt werden, dass sich die Molek{\"u}le der FS in Schichten anordneten, in denen jedes Molek{\"u}l mit vier Nachbarmolek{\"u}len {\"u}ber Wasserstoffbr{\"u}cken verbunden war. Das BGG-K+ kristallisierte als Monohydrat. In dieser Kristallstruktur bildeten die Kaliumkationen in der bc-Ebene mit den BGG-Anionen ober- und unterhalb Schichten. Im Gegensatz zu der FS waren keine intermolekularen Wasserstoffbr{\"u}cken zu beobachten. Die 15N-Festk{\"o}rper-NMR-Spektren des BGG-K+ und der IL zeigten die gleiche chemische Verschiebung f{\"u}r den unsubstituierten Stickstoffes N-1' der Pyrazolgruppe und belegten somit ebenfalls die ionische Struktur der IL im festen Zustand. Die amorphe Struktur der IL wurde mittels R{\"o}ntgen-Pulverdiffraktometrie und Polarisationsmikroskop best{\"a}tigt und eine fl{\"u}ssigkristalline Phase konnte ausgeschlossen werden. Die IL zeigte im Vergleich zu der FS eine 700-fach schnellere Aufl{\"o}sungsrate J und eine signifikante Verl{\"a}ngerung der Dauer der {\"U}bers{\"a}ttigung in w{\"a}ssriger L{\"o}sung. Der sprunghafte Anstieg der Kon-zentration in L{\"o}sung („spring") und die Dauer der {\"U}bers{\"a}ttigung („parachute") wurden mittels photometrischen und potentiometrischen Titrationen untersucht. Mit Hilfe der NMR-Spektroskopie konnte der Mechanismus der {\"U}bers{\"a}ttigung aufgekl{\"a}rt werden. Das sterisch anspruchsvolle Gegenion Tetrabutylphosphonium verhinderte die Protonierung der deprotonierten Sulfonamidgruppe von BGG. In L{\"o}sung kam es zur Bildung von Aggregaten („Cluster"), in die sich das Gegenion teilweise einlagerte. Nach der Protonierung und der Bildung von Kristallisationskeimen pr{\"a}zipitierte die ungeladenen FS und der metastabile Zustand der {\"U}bers{\"a}ttigung („parachute") brach zusammen. Um den Einfluss der Struktur des Gegenions auf die Aufl{\"o}sungsrate und die Dauer der {\"U}bers{\"a}ttigung zu untersuchen, wurden ca. 40 Phosphonium- und Ammonium-Kationen synthetisiert. Die Schmelzpunkte der Phosphonium- und Ammonium-Salze wurden mittels dynamischer Differenzkalorimetrie (DSC) ermittelt. F{\"u}r das Phosphonium-Salz P3332OH-Bromid konnte eine enantiotrope Umwandlung der Modifikationen mittels temperaturabh{\"a}ngiger XRPD-Messungen best{\"a}tigt werden. Die Zelltoxizit{\"a}ts-Untersuchungen der Phosphonium- und Ammonium-Salze an humanen Leberzellen (HepG2), Nierenzellen (HEK 293T) und murinen Makro-phagenzellen (J774.1) zeigten, dass mit h{\"o}herer Lipophilie die Zelltoxizit{\"a}t zunahm. Polare Kationen zeigten keine Zytotoxizit{\"a}t (IC50 > 1000 µM). Die Zelltoxizit{\"a}t der Ammonium-Salze war im direkten Vergleich mit den Phosphonium-Salzen etwas geringer. Die synthetisierten Phosphonium- und Ammonium-Salze, die als Chloride-, Bromide- und Iodide vorlagen, wurden durch Anionenaustausch in Hydroxide umgewandelt. Die Ionischen Fl{\"u}ssigkeiten wurden in einer S{\"a}ure-Base-Reaktion mit der freien S{\"a}ure des BGG-Molek{\"u}ls und den Hydroxiden hergestellt. Der ionische Charakter konnte mittels Bandenverschiebung der deprotonierten Sulfonamidgruppe im IR-Spektrum best{\"a}tigt werden. Die Substanzen waren amorph (XRPD) und die Glas{\"u}bergangstemperaturen (DSC) bewegten sich f{\"u}r die Mono-Kationen im Bereich zwischen 40 °C - 97 °C, f{\"u}r Dikationen 81 °C - 124 °C und f{\"u}r Trikationen 124 °C - 148 °C. Damit erf{\"u}llten einige Substanzen die Definition einer Ionischen Fl{\"u}ssigkeit nicht (Smp. < 100 °C) und wurden daher als Niedrig-Gitter-Enthalpie-Salze (low lattice enthalpy salt = LLES) bezeichnet. Die ILs und LLES zeigten signifikante Unterschiede in der Aufl{\"o}sungsrate J, der {\"U}bers{\"a}ttigungszeit und der Wasserdampfsorption. In dieser Arbeit konnte gezeigt werden, dass allein durch die Auswahl des Gegenions wichtige Parameter f{\"u}r die orale Bioverf{\"u}gbarkeit gesteuert werden k{\"o}nnen. Durch diesen Ansatz war es m{\"o}glich, aus dem sehr schlecht wasserl{\"o}slichen Arzneistoff BGG492 Ionische Fl{\"u}ssigkeiten bzw. LLES herzustellen, die sich drastisch schneller aufl{\"o}sten und teilweise {\"u}ber mehrere Stunden {\"u}bers{\"a}ttigte L{\"o}sungen bildeten. Insgesamt zeigte sich, dass durch eine Zunahme der Polarit{\"a}t des Gegenions eine gr{\"o}ßere Aufl{\"o}sungsrate J und eine geringere Zelltoxizit{\"a}t erzielt werden konnten. Jedoch verringerte sich dadurch die Dauer der {\"U}bers{\"a}ttigung in L{\"o}sung und erh{\"o}hte die Hygroskopizit{\"a}t der ILs und LLES.},
  subject      = {Bioverf{\"u}gbarkeit},
  language  = {de}
}
@phdthesis{Mandel2014,
  author    = {Mandel, Philipp},
  title     = {Entstehung von oxidativen Stressmarkern in DNA und RNA nach der Behandlung mit den Hormonen Angiotensin II und Aldosteron in vitro und in vivo : Vergleich von drei Analysemethoden zum Nachweis von 8-Oxo-2'-desoxyguanosin in LLC-PK1-Zellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111190},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {The detection of oxidative stress markers has gained increasing importancy in the early investigation of diseases like diabetes, cancer or hypertension. 8 oxo 2' deoxyguanosine (8-oxodG) is the main marker, which is used for the intracellular detection of oxidative stress levels. However, the oxidative stress markers 8 oxoguanine (8-oxoGua), a product of the DNA base excision repair and 8 oxoguanosine (8-oxoGuo), a marker for oxidative damaged RNA have received less attention up to now. The renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation processes of the blood pressure system. During hypertension angiotensin II (Ang II) and aldosterone (Aldo) are released in high concentrations over a longer period leading to non-physiological effects of the RAAS hormones. Subsequently, an increase of the intracellular oxidative stress level in kidney cells can be measured. The aim of this thesis is the in vitro and in vivo detection of the oxidative damage in DNA and RNA by measuring oxidative stress markers, especially 8-oxodG which is triggered by Ang II and Aldo. In vitro experiments were carried out in LLC-PK1, a cell line originated from porcine kidney cells. It could been shown that Ang II and Aldo led to a dose-dependent increase of DNA damage in the cells. A time-dependent increase was detected for the first 30 minutes of the treatment. For the rest of the experimental set up (4 h) the level of detected DNA damage remained constant. The FPG comet assay and the immunocytochemical staining showed a significant increase of 8-oxodG in the cells, whereas the HPLC-MS/MS measurement only detected a small increase of 8-oxodG in the DNA. The FPG enzyme, which recognises also other oxidized purines besides 8-oxodG, which led to an overestimation of 8-oxodG in the comet assay. Also, the 8 oxodG antibody, which was used in the immunocytochemical analysis, detected higher amounts of 8-oxodG most likely due to its side reactions with other oxidized DNA structures. One of the main advantages of the last mentioned methods is the direct measurement in damaged cells, whereas the HPLC-MS/MS requires an isolation of the DNA. During this isolation process the oxidative stress markers can be oxidized and the detection can become imprecise. The main purpose of the in vivo experiments was the detection of the oxidative stress marker 8-oxoGua, 8-oxodG and 8-oxoGuo in the urine of test animals. The treatment of C57BL/6 mice and Sprague Dawley (SD) rats with the RAAS hormones led to an increase of the blood pressure, higher DNA damage due to oxidative stress as well as an increased excretion rate of oxidative stress markers. The inhibition of the angiotensin II type 1- or mineralocorticoid receptor and a mutation of the AT1a gene could show, that the DNA damage is independent from the hypertension. In addition, it was shown that the NOX4 is not alone responsible for the oxidative stress. Other NADPH oxidases must contribute to the induction of oxidative stress inside the cell. Moreover, the activation of the Nrf2 pathway has an influence on the effect of Aldo in SD rats. The excretion rate of the oxidative stress markers in the 20 h urine of the treated animals showed how the equilibrium between the DNA repair and the oxidative stress level was changing over time. The measurement of 8-oxoGuo became more and more popular, because up to the fact that 80 \% of the DNA is translated into RNA. Overall, the detection of 8-oxodG and 8-oxoGuo is feasible for monitoring the disease or the healing process, because the measurement is non-invasive. The detection of 8-oxodG and 8-oxoGuo in nucleic acids is a first step into the field of basic research methods, because it reveals a snapshot of the nucleic acid damage in the cell at a specific time point. Usually, there will be an overestimation of the oxidative stress marker resulting from the analytical method. Although, it is possible to detect an underestimation of oxidative stress markers in tissue samples if not all cell types are damaged equally. Therefore, a primary goal should be the detection of a stable oxidation product of guanine to insure a reliable detection strategy and for a better understanding of the equilibrium of DNA oxidation and repair.},
  subject      = {Oxidativer Stress},
  language  = {de}
}
@phdthesis{HafergebZailer2021,
  author    = {Hafer [geb. Zailer], Elina},
  title     = {Tagging - Development of new qNMR methods},
  doi       = {10.25972/OPUS-21958},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-219583},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {High-resolution nuclear magnetic resonance (NMR) spectroscopy is used in structure elucidation and qualitative as well as quantitative examination of product components. Despite the worldwide development of numerous innovative NMR spectroscopic methods, several official methods that analyze specific substances and do not represent a holistic analysis, are still in use for the quality control of drugs, food and chemicals. Thus, counterfeit or contaminated products of inferior quality can be brought onto the market and distributed despite previous quality controls. To prevent this, three NMR spectroscopic methods have been developed within the scope of this work (1) to study the peroxide value in vegetable and animal oils, (2) for the qualitative and quantitative analysis of metal cations and (3) to determine the enantiomeric excess in chiral alcohols. In oil analysis, titration methods are used to determine the bulk quality parameters such as peroxide value, which represents the concentration of peroxides. Titrations show several drawbacks, such as the need of a large amount of sample and solvents, cross reactions and the low robustness. Thus, an alternative NMR spectroscopic method was developed to improve the peroxide analysis by using triphenylphosphine as a derivatization reagent, which reacts with peroxides in a stoichiometric ratio of 1:1 forming triphenylphosphine oxide. In the 1H-31P decoupled NMR spectrum, the signals of the unreacted triphenylphosphine and the reacted triphenylphosphine oxide are detected at 7.4 ppm and 7.8 ppm, respectively. The ratio of the two signals is used for the calculation of the peroxide concentration. 108 oil samples with a peroxide value between 1 meq/kg and 150 meq/kg were examined using the developed method. Oils with a very low peroxide value of less than 3 meq/kg showed a relative standard deviation of 4.9\%, highly oxidized oils with a peroxide value of 150 meq/kg of 0.2\%. The NMR method was demonstrated as a powerful technique for the analysis of vegetable and krill oils. Another 1H NMR spectroscopic method was developed for the qualitative determination of Be2+, Sr2+ and Cd2+, and for the qualitative and quantitative determination of Ca2+, Mg2+, Hg2+, Sn2+, Pb2+ and Zn2+ by using ethylenediamine tetraacetate (EDTA) as complexing agent. EDTA is a hexadentate ligand that forms stable chelate complexes with divalent cations. The known amount of added EDTA and the signal ratio of free and complexed EDTA are used to calculate the concentrations of the divalent cations, which makes the use of an internal standard obsolete. The use of EDTA with Be2+, Sr2+, Cd2+, Ca2+, Mg2+, Hg2+, Sn2+, Pb2+ and Zn2+ result in complexes whose signals are pH-independent, showing cation-specific chemical shifts and couplings in the 1H NMR spectrum that are used for identification and quantification. In the presented NMR method, the limit of quantification of the cations Ca2+, Mg2+, Hg2+, Sn2+, Pb2+, and Zn2+ was determined with 5-22 μg/mL. This method is applicable in the food and drug sectors. The third NMR spectroscopic method introduced an alternative determination of the enantiomer excess (ee) of the chiral alcohols menthol, borneol, 1-phenylethanol and linalool using phosgene as a derivatizing reagent. Phosgene reacts with a chiral alcohol to form carboxylic acid diesters, made of two identical (RR, SS) or two different enantiomers (RS, SR). These two different types of diastereomers can be examined by the difference of their chemical shifts. In the presented method, the integration values of the carbonyl signals in the 13C NMR spectrum are used for the determination of the enantiomer excess. The limit of quantification depends, among others, on the sample and on the non-labelled or 13C-labelled phosgene used for the analysis. In the case of menthol, a quantification limit of ee=99.1\% was determined using non-labelled phosgene and ee=99.9\% using 13C-labelled phosgene. The 13C NMR method was also applied for the quality control of the enantiomeric purity of borneol, 1-phenylethanol and linalool. The developed 13C NMR method represents a powerful alternative to Mosher's reagent for investigating the enantiomeric excess in chiral alcohols. This work demonstrates the variety of possibilities of applications for the quantitative nuclear magnetic resonance spectroscopy in the chemical analysis of drugs, food and chemicals using tagging reactions such as derivatizations and complexations. The nuclear resonance spectroscopic methods developed in this research work represent powerful alternatives to the previously used quality control techniques.},
  subject      = {NMR Spektroskopie},
  language  = {en}
}