Browsing by Subject "HPLC"
Now showing 1 - 7 of 7
Results Per Page
Sort Options
Item 4-Hydroxy-2-Trans-Nonenal (HNE), a toxic degradation product of lipid oxidation, in fried chicken and donut(2017-08) Yin, NingAlpha, beta-unsaturated-4-hydroxyaldehydes, a group of secondary lipid oxidation products, are highly interesting due to their high reactivity to various biological compounds including amino acids and DNA. 4-Hydroxy-2-trans-Nonenal (HNE), one of the most abundant and toxic compound in this group, was measured in commercial fried chicken breasts, chicken thighs, chicken nuggets and raised glazed donuts from different fast food stores and supermarkets. Samples were analyzed for fat content, fatty acids distribution and HNE concentration. Fried chickens and donuts were selected because they are very common fast food and they are deep-frying at high temperatures (160-190 °C). Preliminary experiments were conducted using the thiobarbituric acid (TBARS) assay to obtain the secondary lipid oxidation products such as aldehydes, ketones and related carbonyl compounds. HNE concentration was measured as 2,4-dinitrophenylhydrazone derivatives using an HPLC method. HNE concentrations in fried chickens from 3 different sources are between 10.02 and 11.89 µg HNE/100 g breast and between 19.80 and 24.41 µg HNE/100 g thigh. For chicken nuggets and popcorn chicken, HNE concentrations are between 9.00 and 47.93 µg HNE/100 g samples. For donut samples from 3 different brands, HNE concentrations were between 18.55 and 21.71 µg HNE/100 g donut. Measured HNE amount in chicken thighs samples is greater than chicken breasts samples, suggesting the heme iron content existed in dark meat possibly act as a catalyst to accelerate the lipid oxidation in the meat. These results indicated that HNE is incorporated into the fried chickens and glazed donuts samples from frying oils and might pose a public health concern for long-term consumption given the toxicity of HNE.Item Bacterial Mucin Degradation in the Cystic Fibrosis Airways: A Potential Regulator of P. aeruginosa Pathogenicity.(2023-05) Villarreal, AlexCystic fibrosis (CF) is characterized by impaired mucociliary clearance, leading to mucus accumulation and chronic bacterial respiratory infections. Pseudomonas aeruginosa (PA) is a canonical pathogen in these infections and is found to nutritionally benefit from cross-feeding interactions with co-colonizing mucin-degrading bacterial genera. However, the impact of these cross-feeding interactions on the regulation of PA virulence remains unclear. Implementing a reductionist experimental approach, this thesis aims to investigate the proposed cross-feeding model to identify key exchanged metabolites and elucidate mechanisms underlying PA virulence. To successfully isolate and analyze variables of the multifaceted cross-feeding model, several specialized experimental tools and techniques were developed and described in Chapter 2. These tools were employed in Chapter 3 to fully characterize the growth profiles of several individual representative mucin-degrading bacterial species and investigate the effects of their secondary metabolites on PA physiology. Chapter 4 builds upon this by characterizing complex mucin-degrading bacterial communities enriched from clinical CF sputum samples, and again investigating their effects on PA physiology in vitro. These data establish validated methods for the field of CF research, expand our understanding of PA physiology, and pave the path for future development of novel targeted CF therapeutic strategies.Item Charge-Enhanced Brønsted Acids: Catalyst Designs and Analytical Methods(2020-06) Payne, CurtisHydrogen bonding is a crucial interaction in Nature and plays an important role in the structure and stability of DNA, ion molecular recognition, and the reactivity of enzymes. In the last few decades, synthetic chemists have investigated hydrogen bond donors in the development of metal-free catalysts for a variety of reactions. In these studies, a more acidic species generally results in improved reactivity and stereoselectivity, and a recent investigation uncovered that the acidity of a compound in nonpolar media correlates with its gas phase acidity better than its pKa value in DMSO. As a result, charged hydrogen bond donors and Brønsted acids have been synthesized and are more effective catalysts in nonpolar media compared to their neutral analogues. In this dissertation, this charge enhanced effect is examined through a structure-reactivity relationship and new organocatalytic designs are explored. Additionally, improved analytical methods for the characterization and investigation of these cationic Brønsted acids are discussed.Item Chromatographic selectivity and hyper-crosslinked liquid chromatography stationary phases.(2010-01) Zhang, YuThe development of new stationary phases have always been of great interest in HPLC and has become increasingly important in recent years mainly driven by rapidly evolving industrial need as well as the quest for higher throughput HPLC analyses for better resolution and higher sensitivity. In this thesis, we developed a family of silica based RPLC stationary phases based on a novel hyper-crosslinked (HC) platform, prepared through a multi-layer, two-dimensional, orthogonal polymerization reaction. The resulting stationary phases showed better stability, higher efficiency and novel selectivities, which are the three essentials properties of the stationary phase that users are looking for and column developers strive to achieve. We first studied the synthesis and full characterization of a novel mixed-mode reversed-phase/weak cation exchange (RP/WCE) phase by introducing a small amount of carboxylate functionality into a hydrophobic hyper-crosslinked (HC) platform. The phase thus prepared shows a mixed-mode retention mechanism, allowing for both neutral organic compounds and charged bases to be separated simultaneously on the same phase under the same conditions. More importantly, the inherent weak cation exchange groups allow simple mobile phases to be used thereby avoiding the mass spectrometric ionization suppression problems concomitant to the use of non-volatile additives such as strong amine modifiers (e.g. triethylamine) to elute basic solutes from the strong cation exchange phases or ion pairing reagents (e.g. trifluoroacetic acid, ClO4-) to retain these solutes on conventional ODS phases. We next studied the development of a highly hydrophilic HC-OH phase prepared by hydrolyzing residual benzyl chloride groups on the hydrophobic platform. This phase is potentially useful as a candidate for use as the first dimension phase of comprehensive two-dimensional LC where column stability and low retentivity are greatly desired. We also developed a novel graphical method, the phase selectivity triangle plots, for visualizing the effect of surface chemistry (e.g. C18 vs. Phenyl vs. Fluoro) on stationary phase selectivities. The use of the new plots assists the selection of appropriate stationary phases for method development in both isocratic and gradient elution.Item Mass spectrometry-based analysis of urinary metabolites of 1,3-Butadiene (BD) in humans and influence of BD-DNA adducts on DNA replication(2013-10) Kotapati, SrikanthCigarette smoking is a known risk factor for the development of lung cancer: approximately 1 out of 5 heavy smokers will develop the disease. However, there are significant differences in risk of lung cancer among smokers from different ethnic/racial groups. African American and Native Hawaiian smokers are at a higher risk of lung cancer than European American, Japanese American or Latin American smokers. Cigarette smoke has more than 70 known carcinogens. Following metabolic activation to electrophilic species, these carcinogens can form covalent DNA adducts, which are capable of inducing heritable mutations ultimately resulting in lung cancer. It has been hypothesized that the observed ethnic/racial differences in lung cancer risk in smokers are due to different frequencies of specific polymorphisms in drug metabolizing genes, leading to a different degree of carcinogen bioactivation to DNA-reactive intermediates. 1,3-Butadiene (BD) is among the most abundant and potent carcinogens present in cigarette smoke. BD is metabolically activated primarily by CYP2E1 to form 3,4-epoxy-1-butene (EB), hydroxymethyl vinylketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). EB, HMVK, EBD, and DEB have been shown to modify DNA bases to form promutagenic adducts. Alternatively, EB, EBD, and DEB can undergo detoxification via epoxide hydrolysis (the main pathway in humans) or glutathione conjugation and further metabolic conversion into urinary mercapturic acids, 1-hydroxy 2-(N-acetylcysteinyl)-3-butene (MHBMA), 1,2-dihydroxy-4-(N-acetyl cysteinyl)-butane (DHBMA), 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA), respectively. The research presented in this thesis focuses on revealing any ethnic/racial differences in metabolism of BD in smokers and examining the ability of BD-DNA adducts to cause mutations. In the first part of the thesis, we have identified two novel metabolites of BD which have not been previously detected in vivo: 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA). To enable their detection in smokers, sensitive and specific HPLC-ESI--MS/MS methods were developed for both metabolites in human urine. We observed significant amounts of THBMA in samples from smokers, non-smokers and occupationally exposed workers. In contrast, bis-BDMA amounts in urine of smokers and occupationally exposed workers were below the method's limit of detection, although it was found in urine of F344 rats exposed to 62.5 or 200 ppm BD. Additionally we found significant interspecies differences in BD metabolism between laboratory rats and humans. DHBMA accounted for only 47% of BD urinary mercapturic acids in rats while the corresponding percentage in humans is 93%. We further developed a high throughput HPLC-ESI--MS/MS method for the quantification of MHBMA and DHBMA in humans and applied this method to quantify urinary BD-mercapturic acids metabolites in workers from a BD and styrene butadiene rubber (SBR) manufacturing facility and smokers belonging to different ethnic groups in two separate multi-ethnic cohort studies. Workers occupationally exposed to BD excreted significantly more BD-mercapturic acids than administrative workers at the same plant. In a small multi-ethnic study of smokers belonging to European American, Native Hawaiian and Japanese American (N = 200 per group), mean urinary MHBMA and MHBMA/DHBMA+MHBMA metabolic ratio were highest in European American and lowest in Japanese American smokers. Similar results were obtained in the larger study (N = 450 per group) composed of European American and African American smokers. Urine of European American smokers contained higher concentrations of MHBMA than that of African Americans. Genome-wide association study (GWAS) analysis conducted for the larger multi-ethnic group has revealed significant associations between single-nucleotide polymorphisms (SNPs) in chromosome 22 (22564172bp - 22735492 bp, nearby genes GSTT1, GSTT2, DDT and MIF) and urinary BD-mercapturic acid levels in smokers, providing the first evidence for genetic and ethnic/racial differences in metabolism of BD.The second part of my thesis work has focused on evaluating the mutagenic ability of three recently discovered BD-dA lesions: N6-(2-hydroxy-3-buten-1-yl)-adenine (N6-HB-dA), N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA), and 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-&gamma-HMHP-dA). In vitro translesion synthesis experiments were performed on synthetic oligonucleotides containing each of the three lesions at a site-specific position by gel electrophoresis and HPLC-MS/MS. We found that human translesion synthesis (TLS) polymerases hPols &eta, &kappa, &iota and human polymerase &beta were able to bypass (S)-N6-HB-dA in an error-free manner because of the conserved Watson-Crick base pairing with dT. However, replication past both (R,R)-N6,N6-DHB-dA and (R,S)-1,N6-&gamma-HMHP-dA lesions by TLS polymerases hPols &eta and &kappa was highly error-prone, resulting in A&rarrT, A&rarrC mutations and frameshift deletions. This is the first study that identifies (R,R)-N6,N6-DHB-dA and (R,S)-1,N6-&gamma-HMHP-dA as BD-DNA adducts potentially responsible for the induction of A*rarrT mutations by BD.Item Production of Astaxanthin by Haematococcus Pluvialis Using Deproteinized Whey Permeate as a Nutrient Source(2021-03) Brandt, MadelineThe goal of this project was to demonstrate the feasibility of a value-added use for deproteinized whey permeate (DWP) as feed material for the algae Haematococcus pluvialis to produce astaxanthin, the main carotenoid contributing to salmon and shrimp pigmentation. Algae was grown under 3 mixotrophic conditions: standard WC medium, standard WC medium with 10 g/L DWP, and 10 g/L DWP alone. Algae were harvested and carotenoids extracted with a modified Mojonnier method. The extracts contained free astaxanthin and astaxanthin mono- and diesters. Extracts were treated with cholesterol esterase to facilitate total astaxanthin quantification via HPLC-UV/VIS-MS/MS. Total astaxanthin dry weight was 2.11 μg (SD=0.80), 5.98 μg (SD=1.79), and 9.97 μg (SD=3.74) for the three treatments, respectively (n=4), indicating that DWP supports growth of H. pluvialis and that the algae can utilize lactose in DWP without hydrolysis. This is promising for dairy processors, who could utilize DWP as a novel income stream.Item Selectivity Optimization in Tandem Column Liquid Chromatography Using the Eluent Composition as the Tuning Variable(2018-09) Bigert, MatthewClearly, in typical separations in HPLC, selectivity is the most important variable for improving resolution and the optimization of selectivity is the primary focus of most method development effort in reversed-phase liquid chromatography (RPLC). We have developed a family of novel techniques in which two columns with markedly different chromatographic selectivities are combined in tandem. One then uses another chromatographic variable (e.g. temperature, flow rate, eluent composition) which has different values for the two columns to continuously “tune” the selectivity. We call this family of techniques XT2C, meaning that the variable X (temperature, etc.) is used to continuously tune the combined selectivity of the tandem columns. By serially coupling the columns, our approach eliminates the common “selectivity discontinuity” problem encountered by analysts when the type of column used is changed. Previously, we utilized the thermally tuned tandem column (T3C) concept in which selectivity was adjusted by independently tuning the two column temperatures. The primary difference between the experimental setup for T3C and XT2C is that a second pump and static micro-mixer are placed between the two columns. In this study, we describe the eluent tuned tandem column concept in which selectivity is continuously tuned by independently controlling the eluent composition of each column. When the percent organic modifier in the eluent (%B) is adjusted, the concept is denoted “solvent tuned tandem column (ST2C)”. Likewise, when the ionic strength (buffer concentration) is tuned, the concept is referred to as ionic strength tuned tandem column (IT2C). The objective of the present study is to evaluate the applicability of IT2C and ST2C to mixtures of ionic and non-ionic analytes, respectively. We also describe the use of a simple computer assisted optimization strategy based on the window diagram method. This strategy allows for XT2C optimization based on only four to six initial data acquisition runs. ST2C and IT2C are comparable in terms of chromatographic performance to T3C and provide flexibility in optimizing selectivity but do not require thermally stable columns.