The aim of this diploma thesis was to develop a systematic procedure for the development of HPLC methods using the design of experiments. The system was developed based on the development of three HPLC methods for the determination of the purity of active substances using the design of experiments approach. The HPLC method for determining the purity of esomeprazole was selected to develop the systematic process for the development of HPLC methods by statistical approach. Experimental space was explored to find more suitable separation conditions. The second method used to develop the systematic procedure was the method for determining the purity of bisoprolol. This method was converted to a column of smaller size and the composition of the aqueous part of mobile phase was modified compared with the original pharmacopoeial method. Experimental space was explored to find more suitable separation conditions using the design of experiments. Last the method for determining the purity of risperidone was chosen. The composition of the aqueous part of mobile phase was changed in contrast to the original pharmacopoeial method. Experimental space was explored to find more suitable separation conditions using the design of experiments. For all studied HPLC methods, the values of the monitored chromatographic...

Presented diploma thesis is focused on production of secondary metabolites under influence of external factors.in cannabis (Cannabis sativa L.). Theoretival part contains phylogenetical, morphological and botanical characterization of the given species. The part is further expanded by description of major cannabis constituents and their biosynthetic pathways. In the end of the chapter, methods for elicitation are described. Main goal of the experimental part was to compare influence of vaious fertilizers and product containing coloid silver on morphological characteristics and selected secondary metabolites content. Highest yield was observed within plants fertilized by Powder Feeding. Highest THC content was observed in plants fertilized by Jungle in da Box. Variation of height, yield and secondary metabolites within experimental population was observed, however was not statistically conclusive. Influence of Altron Silver was not statistically proven.

The thesis objective was to check out the effect of nitrogen fertilizers and nitrogen fertilizers with sulphur in nutrition of spring barley (variety Bojos) and winter wheat (variety Mulan). The quality of protein complex was determined on grain samples by the HPLC method. The application of sulphur showed also in the fractional composition of the proteinous complex. The wheat grain without applied sulphur fertilizers had significantly higher percentage proportion of ?-gliadins, albumins and globulins compared to the variants treated this way. It was at the expense of gluten proteins. The greatest fertilization effect was proved after application of Yara Vita Thiotrac in late vegetation period. The proportion of barley´s LMW glutelins, D-hordeins, albumins and globulins has demonstrably increased in the variants with applied sulphur. The changes of fractional protein composition caused by degradation of high-molecular compounds during malting and mashing were confirmed.

The thesis entitled Screening of Polyphenols in Coffee deals with origins and attri-butes of particular types and technologies of coffee making.The theoretical section focuses on occurance and importance of biologically active compounds contained in coffee that might have both positive and negative impact on human´s health. I also mention the legislation related to the issue of defining and labeling the coffee. Regarding the most current lifestyle diseases and various social groups I describe the general influence of coffee on human´s health and the most common methods of determination polyphenols in food.In the practical part I use the High-performance liquid chromatography to identify the spectrum and amount polyphenolic com-pounds at selected samples of coffee of various geographical origin. Overall I tested samples of 10 roasted and 8 unroasted Arabica coffee and 2 samples of Robusta coffee.

Purpose: Use of ampicillin in outpatient parenteral antimicrobial therapy (OPAT) has historically been complicated by frequent dosing and short beyond use dates. However historic stability data relied on inaccurate testing methods. The purpose of this study is to evaluate the stability of ampicillin using high-pressure liquid chromatography (HPLC), the gold standard, in a real-world OPAT dosing model using continuous infusion at room temperature over 24 hours immediately following preparation compared to batches stored under refrigeration for 24 hours, 72 hours, and 7 days. Methods: An HPLC method was developed and validated as stability – indicating according to guidance in USP general Chapter . Method development included linearity, precision, accuracy, repeatability and forced degradation. Four batches were prepared using 4 different lots from 2 different manufacturers for each storage condition (immediate, 24 hours, 72 hours, and 7 days). Three 2-gram vials were each reconstituted with 10 mL of sterile water for injection (SWFI) and added to 250 mL of normal saline by a licensed pharmacist and stored in a laboratory refrigerator (2 – 8oC). A pump system was used to continuously circulate the solutions through medical grade tubing at room temperature. One milliliter aliquots were removed from each batch at time 0, 4 hours, 8 hours, 12 hours and 24 hours and analyzed for ampicillin concentration using the aforementioned HPLC method. The samples were filtered prior to analysis using a 0.22-micron syringe filter and analyzed in triplicates along with freshly prepared calibration samples (24 – 12 mg/mL). Peak area was used to determine percent recovery for each sample. Results: Each batch was assayed for initial concentration (20.34 – 21.50 mg/mL) upon preparation, and percent recovery was compared to that initial concentration thereafter. Acceptable recovery was defined as 90 – 110% of initial concentration. On the day of product preparation (immediate use), the average percent recovery over 24 hours was 96.4%. The other average percent recoveries were as follows: 95.8% (24-hour storage), 94.6% (72-hour storage) and 90.3% (7-day storage). These data represent the average percent recovery for all time points during the 24 hours sampling (n = 60 for each experiment). When evaluating individual time points, the percent recovery remained above 90% for all batches and time points except for the 7-day storage experiment. Under 7-day storage conditions, the percent recovery fell below 90% after 4 hours of circulation through the medical grade tubing. Furthermore, 95% confidence interval for percent recovery for ampicillin in the samples stayed within 90 – 110% of the initial concentration for the duration of the experiment for all test groups except 7-day storage. Conclusions and Relevance: Ampicillin can be prepared and stored in a refrigerator for up to 72-hours prior to continuously infusing at room temperature over 24 hours with less than a 10% loss of potency over the dosing period. This model supports twice weekly OPAT delivery of ampicillin.

The thesis is dedicated to the phospholipids and their polar headgroups analysis by gas chromatography, capillary electrophoresis and mass spectrometry. Phospholipids are the most important polar lipids and they are classified into phospholipid classes according to their phosphorylated groups. Phospholipids can be found in cell membranes and the changes in their ratio are monitored to research the impact of external conditions on cells. Actually thin layer chromatography is still used for phospholipid class ratio analyses. It is not suitable for microbiological research due to its time demandingness. The presented compendium of papers engaged in phospholipid classification is targeted on Bacillus subtilis strain, which produces potential antibiotics with detergent effect - surfactin. Published methods can be used for research of optimal conditions for producing microbe cultivation. Because non-polar parts of the phospholipid molecule (fatty acids) can affect the analysis methods on spliced polar headgroups have to be designed. Capillary electrophoresis and gas chromatography methods were developed and the latter one was further optimized for simultaneous analysis with fatty acids. Additional part deals with an alternative approach which consists in direct injection on mass spectrometer of intact...

Introduction. N-acetylcysteine (NAC) is the antidote for acetaminophen (Tylenol) toxicity from over ingestion leading to 56,000 emergency room visits yearly. This is worrisome due to the risk of hepatoxicity, especially in children and adolescents. Often, nausea and vomiting are associated with NAC use and is treated acutely by ondansetron (Zofran), a 5-HT3 receptor antagonist. Inconveniently, the NAC 21-hour intravenous (IV) infusion needs to be halted with IV flushing before ondansetron can be administered. Another IV flushing follows before NAC is resumed. This causes treatment interruption in a medical emergency; therefore, we are investigating the IV compatibility of NAC and ondansetron to reduce the steps in treating acute nausea/vomiting. Methods. A reverse phase high-performance liquid chromatography (RP-HPLC) method was utilized for NAC quantification. The analysis was conducted on an Agilent Eclpise XDB-C18 column (3.5 micron, 4.6 x 150 mm) with a mobile phase containing acetonitrile (ACN), water (10:90 v/v), and 0.1 % trifluoroacetic acid (TFA). The flow rate was set at 0.500 mL/min with an injection volume of 10 microliters and a temperature of 50oC. A UV wavelength of 212 nm was utilized for detection of NAC. A liquid chromatography mass spectrometry (LC – MS/MS) method was able to quantify levels of ondansetron. A Waters XBridge C18 column (3.5 micron, 4.6 x 150 mm) was used for separation of ondansetron. The mobile phase included ammonium formate buffer (pH 3.0, 5 mM) and acetonitrile (15:85, v/v) with the flow rate set at 0.500 mL/min. Electrospray ionization interface is set in the positive mode for measurement of ondansetron using a precursor ion of m/z 294.0200. Results. The HPLC-UV and LC-MS/MS methods for NAC and ondansetron, respectively, will be validated for linearity, precision and accuracy. Then the methods will be applied toward a chemical compatibility investigation of NAC and ondansetron through medical grade tubing and y-site. The ideal outcome would be to confidently assume NAC and ondansetron are IV compatible for y-site administration to avoid infusion interruption for treatment of acetaminophen toxicity. Conclusion. IV compatibility for NAC and ondansetron affords no infusion interruptions reducing unnecessary risk of acetaminophen toxicity. This also decreases risk of medical errors based on the multi-step process to administer ondansetron with receiving NAC. Overall, compatibility could create safer, more efficient protocols for treatment of acute nausea/vomiting from NAC administration.

Background: Aceclofenac is a phenylacetic acid derivative with analgesic and anti-inflammatory properties and an improved gastrointestinal tolerance compared with other NSAIDs, such as diclofenac. Objective: This study was conducted to compare the bioavailability of 2 branded formulations of aceclofenac 100 mg (test and reference) marketed in Korea. Methods: This single-dose, randomized, open-label, 2-period crossover study in healthy Korean adult volunteers was conducted at Hanyang University Medical Center (Seoul, Republic of Korea). Subjects received 1 tablet of each aceclofenac 100-mg formulation. Study drugs were administered with 240 mL of water after a 10-hour overnight fast on each of 2 treatment days separated by a 1-week washout period. After study drug administration, serial blood samples were collected over a period of 12 hours. Plasma was analyzed for aceclofenac concentration using a validated high-performance liquid chromatography method with visible detection in the range of 0.1 to 20 μg/mL, with a lower limit of quantitation of 0.1 μg/mL. Several pharmacokinetic (PK) parameters, including Cmax, Tmax, t1/2, AUC0-t, AUC0-∞, and ke, were determined from the plasma concentrations of the 2 aceclofenac formulations. Cmax, AUC0-t, and AUC0-∞ were used to test for bioequivalence after log-transformation of plasma data. The predetermined, regulatory range of 90% CI for bioequivalence was 0.80 to 1.25. Results: A total of 24 subjects were enrolled (20 men, 4 women; mean [SD] age, 23.5 [1.4] years; mean [SD] weight, 68.1 [11.5] kg). No significant differences were found based on analysis of variance, with mean values and 90% CIs of test/reference ratios for these parameters as follows: Cmax, 10.57 versus 9.79 μg/mL (0.961-1.225); AUC0-t, 19.95 versus 19.93 μg · h/mL (0.937-1.037); and AUC0-∞, 20.75 versus 20.48 μg · h/mL (0.949-1.049). Conclusion: In these healthy Korean volunteers, results from the PK analysis suggested that the test and reference formulations of aceclofenac 100-mg tablets were bioequivalent, based on the regulatory definition.

Polymer additives exemplify many classes of compounds which possess a wide variety of chemical (i.e., phenols, amides, esters) and physical (i.e., volatility, solubility) properties. They are incorporated into polyolefins and other such polymeric materials for a number reasons: (a) to prevent degradation by ultraviolet light, heat, and oxygen; (b) to aid in the processing of the polymer; and (c) to modify the physical properties of the polymer. Since the purity and amount of additive can affect polymer properties, it is very important to characterize and quantify additives in polymer products. Traditional liquid solvent/polymer extraction methods, which involve dissolution/precipitation, are time-consuming, uneconomical, and the recoveries are significantly lower than 90%. In recent years, analysis with supercritical fluids (SFs) has emerged as an alternative analytical technique because SFs afford higher diffusivity and lower viscosity. In this research, an on-line Supercritical Fluid Extraction (SFE)/Supercritical Fluid Chromatography (SFC) system was assembled to provide efficient extraction and separation of polymer additives with quantitative results. The effects of various SFE/SFC parameters, such as trapping temperature, injection temperature, extraction pressure and temperature, dynamic extraction time, and fluid flow rate on extraction and separation efficiencies of different additive standards (i.e., BHT, BHEB, Isonox 129, Irganox 1076 and Irganox 1010) were investigated. Optimized conditions were employed to quantitatively extract additives from LDPE. Identification of additives was performed by comparing the retention time with each additive standard. Results obtained from on-line SFE/SFC were compared to results from off-line SFE/High Performance Liquid Chromatography (HPLC) and off-line Enhanced Solvent Extraction (ESE)/HPLC. / Master of Science

Student Number : 0009756Y - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / A versatile nitrile-degrading bacterium was isolated through enrichment culturing of soil samples from Johannesburg, South Africa. It was identified as Rhodococcus rhodochrous and submitted to the ATCC culture collection as strain BAA-870. This organism was determined to be a potential biocatalyst in that it contains a two enzyme system with strong nitrile-converting activity comprising nitrile hydratase and amidase. The development of a suitable assay for measuring the activity of the enzymes of interest was explored. A pHsensitive indicator-based assay was found to be suitable only for colorimetrically identifying highly concentrated enzymes with acid-forming activity. An ophthaldialdehyde- based fluorimetric assay was found to be applicable to conversions of select compounds, but the assay could not be used to measure the activity of Rhodoccocus rhodochrous ATCC BAA-870. High performance liquid chromatography was the most suitable method for reliable and quantitative measurement of nitrile hydrolysis, and is applicable to monitoring activities of whole-cell and cell-free extracts. Initial analysis of six compounds, benzonitrile, benzamide, benzoic acid, hydrocinnamonitrile, 3-hydroxy-3- phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid, was performed by HPLC to measure linearly the average retention area, amount and absorbance of the compounds up to 10 mM concentrations. The conversion of the substrates benzonitrile, benzamide and 3- hydroxy-3-phenylpropionitrile were further analysed with respect to time and enzyme concentration. Conversion of benzonitrile to benzamide by the nitrile hydratase was rapid and could be measured in 10 minutes. Conversion of benzamide to benzoic acid by the amidase was considered the rate-limiting step and could be followed for 90 minutes of the reaction at the concentrations tested. Conversion of 3-hydroxy-3-phenylpropionitrile was linearly measured over 20 minutes. Mass spectral analysis was used to confirm, at a structural level, relatively less volatile reactant compounds with a higher thermal stability, including benzamide, 3-hydroxy-3-phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid. Protein concentration studies indicated that activity against benzonitrile was probably due to a nitrile hydratase with potent activity rather than a concentrated enzyme, since formation of benzamide from benzonitrile showed first order reaction kinetics at protein concentrations less than 0.2 mg/ml. Formation of benzoic acid from benzamide was linear up to 1.3 mg total protein and product formation from 3-hydroxy-3-phenylpropionitrile was linear up to 1.4 mg total protein. Overlapping activities against benzonitrile and 3- hydroxy-3-phenylpropionitrile indicate that the nitrile hydratase has differing substrate specificity for the two compounds, with higher activity toward the small aromatic mononitrile, benzonitrile, than the arylaliphatic b-hydroxy nitrile, 3-hydroxy-3- phenylpropionitrile. The nitrile-converting activity of Rhodococcus rhodochrous ATCC BAA-870 would be suitable for biocatalysis as the conversions take place under a wide pH range, require low concentrations of enzyme and reactions are fast. Separation of nitrileconverting activities in Rhodococcus rhodochrous ATCC BAA-870 was undertaken using various chromatography methods to establish a simple, one-step protocol for biocatalytic enzyme preparations. HPLC was not suited to assaying nitrile-converting activity in chromatofocusing fractions, and chromatofocusing Ampholyte buffers were found to interfere with activity measurements. Gel exclusion chromatography of the soluble protein extract from Rhodococcus rhodochrous ATCC BAA-870 indicated the enzyme/s responsible for nitrile hydratase activity are high molecular weight proteins ranging from 40 to 700 kDa in size, while the amidase native enzyme is proposed to be roughly 17 to 25 kDa. SDS-PAGE analysis of gel exclusion and ion exchange chromatography fractions indicated nitrile converting activity in Rhodococcus rhodochrous ATCC BAA-870 is likely due to multimer-forming enzymes made up of 84, 56, 48 and 21 kDa subunits. It is postulated that nitrile hydratase is made up of ab and a2b2 tetramers that may form larger enzyme aggregates. Ion exchange chromatography was used to separate nitrile hydratase with high activity against benzonitrile and 3-hydroxy-3-phenylpropionitrile from amidase activity, and showed that an additional, substrate specific nitrile hydratase may exist in the organism.