الفهرس 
General Introduction and Literature ReviewOnly 14 pages are availabe for public view
 |  | from | 328 |  |  |
| | | from | 328 | | |
Abstract
This thesis consists of Four parts:
Part I: General Introduction and Literature Review
Section I.A: Insights from the worldwide obesity epidemic and the current regulatory situation of weight loss dietary supplements
This section involves a general introduction on how obesity is defined and its worldwide prevalence, especially in Egypt. It also deals with obesity pathogenesis including energy homeostasis, adipocyte biology and life cycle, dietary lipid digestion, absorption and metabolism. We highlighted the use of over-the-counter dietary supplements as an effortless option for losing weight. We included a brief overview of the current regulatory situation of dietary supplement products, including the Dietary Supplement Health and Education Act and the efforts of Office of Dietary Supplements – National Institute of Health to set a strict quality control policy for their release to the market.
Section I.B: Drugs under study and literature review
It includes a detailed review on the studied drugs, 2-Phenethylamine HCl, p-Synephrine, p-Hordenine, p-Octopamine, Raspberry ketone and trans-Resveratrol. This review explains chemical and physical properties, natural occurrence, pharmacology, weight loss mechanism of action, pharmacokinetics, safety, situation from official regulatory efforts and different analytical methods reported for detection and quantitation of the studied drugs in literature.
Part II: Potentiometric methods
Section II.A: Preparation and characterization of silver doped copper oxide nanoparticles
This section involves the preparation of silver doped copper oxide (Ag.CuO) nanoparticles using a simple wet co-precipitation synthesis procedure. The fabricated nanoparticles were characterized using Fourier transform – Infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray analysis and UV-Visible spectrophotometry. characterization results showed that these nanoparticles are crystalline irregular spherical shaped with an average particle size of 3.44 ± 0.66 nm. The calculated bandgap energy was 2.85 eV, which confirms their good conductivity. Thus, the synthesized Ag.CuO nanoparticles can be regarded as good conductive sensing interface for electrochemical applications.
Section II.B: Potentiometric assay of 2-Phenethylamine HCl using a novel Silver doped copper oxide / Calixarene composite – glassy carbon-based sensor
In this section, 2-Phenethylamine HCl was assayed for the first-time using ion-selective potentiometric membrane sensors. The three proposed sensors were based on incorporation of sodium tetraphenylborate ion exchanger alone or combined with calix[8]arene or with a novel hybrid composite of the previously synthesized Ag.CuO nanoparticles and calix[8]arene in sensing membranes mounted on a conventional solid contact glassy carbon electrode for sensors 1, 2 and 3; respectively. Nernstian slopes were achieved with slope values of 52.28, 54.64 and 55.34 mV/concentration decade and LOD of 9.88 × 10-5, 7.18 × 10-5, and 9.77 × 10-6 M for sensors 1, 2 and 3; respectively. The proposed sensors showed excellent percent recoveries when applied for the analysis of 2-Phenethylamine HCl in its multi-ingredient formulation and showed no statistical difference with another published NMR spectroscopic method. Among all results, it was found that Ag.CuO / Calixarene combination in the PVC sensing membrane provided the best sensitivity and the most stable and fastest response among all tested sensors.
Part III: chromatographic methods
Section III.A: Dietary Supplement Mislabelling: Case study on selected slimming products by developing a green isocratic HPLC method for their quality control
As validated analytical methods are urgently needed to guarantee the safety and efficacy of dietary supplement products, we present in this section a simple HPLC-PDA method for the quantitation of seven popular slimming ingredients. Studied compounds were Caffeine, Raspberry Ketone, trans-Resveratrol, p-Synephrine, p-Octopamine, p-Hordenine and 2-Phenethylamine. After optimization, separation was carried out on a C18 column and mobile phase was a mixture of acetonitrile: water containing 0.1% phosphoric acid (50:50, v/v). The last compound was eluted at 9.76 min. Separation was efficient showing baseline- separated symmetric peaks, without using any gradient programs, organic mobile phase modifiers or modified stationary phases. Method validation was done following ICH guidelines. Calibration curves were linear over wide concentration ranges and calculated LOD values were in the range 0.02-0.09 µg/mL. Method greenness was assessed using Analytical Eco-scale, GAPI and AGREE metric tools. Further, four random sample products purchased from online supplement stores were assayed. Results proved some mislabeling actions. To support our findings, standard addition was carried out and average % recoveries were in the range 96.67 – 101.44%.
Section III.B: A validated UHPLC-MS/MS method for the determination of free Raspberry Ketone and its alcoholic metabolite in urine: Application to real human samples.
As per the various health benefits of raspberry ketone and that no human clinical pharmacokinetic data is reported in literature, we present for the first time in this work a validated UHPLC/MS-MS method for the simultaneous determination of free Raspberry ketone and its major metabolite, Raspberry alcohol, excreted in human urine. chromatographic separation was done on a C18 column (150 × 2.1 mm, 1.9 μm) with isocratic elution using a mixture of 0.1% Formic acid in water and 0.1% Formic acid in acetonitrile (70:30, v/v) at a flow rate 0.25 mL/min. Detection was performed using multiple-reaction monitoring mode (MRM) in the Electrospray ionization (ESI) positive mode, by monitoring the transitions 107/77 and 107/51 m/z for both compounds. It’s important to note that both tested drugs showed identical MRM profiles and were separated by chromatography. A simple liquid- liquid extraction method was employed for sample preparation using ethyl acetate. Validation was done in accordance with FDA bioanalytical validation guidelines. Linearity range was 20 – 1000 ng/mL for both drugs, which allowed their sensitive determination in real urine samples obtained from healthy human volunteers after oral administration of Raspberry ketone capsules. Analysis of authentic samples showed that Raspberry ketone and its reduced alcoholic derivative are rapidly excreted in urine within only 1 hr. of oral administration and that the administered drug was more abundant than its metabolite among all tested samples. The obtained method is simple, sensitive, accurate, precise, and shows efficient sample preparation, thus, can be adopted for further clinical applications.
Part IV: Spectroscopic methods
Section IV.A: An economic green synchronous spectrofluorimetric assay of Raspberry ketone and its application to Weight Variation testing
We proposed the first native spectrofluorimetric quality control assay of Raspberry ketone. This work relied on the constant wavelength synchronous scan of the Raspberry Ketone native fluorescence, overcoming the demerits of conventional excitation/ emission spectra. For the best measurement conditions, several parameters were optimized including Δλ value, diluting solvent, medium pH and the effect of surfactants/ macromolecules. In aqueous medium (Δλ = 110 nm), a linear relationship was established between synchronous fluorescence intensity at peak maximum 405.6 nm and solution concentration in the range 300-1500 ng/mL. Method sensitivity was recorded with LOD and LOQ values of 60.63 and 183.72 ng/mL; respectively. Validation was done in accordance with International Conference on Harmonization (ICH) guidelines. This validated procedure was then successfully applied to the analysis of Raspberry Ketone in commercially available dietary supplement capsules with average recovery 98.67% ± 1.74 and further extended to weight variation testing following the official United States Pharmacopeial (USP) guidelines. Finally, green assessment was done using Analytical Eco-scale, GAPI and AGREE metric tools and all proved the greenness of our proposal. Our proposed method was simple, eco-friendly, and cheap. It can be conveniently adopted for routine quality control practices especially in developing countries.
Section IV.B: One-pot synthesis of N-doped carbon dots from microwave irradiated egg-white: Application to Raspberry ketone assay by photo-induced charge transfer fluorescence sensing
In this section, we designed one-step economic eco-harmonious microwave-assisted procedure to prepare Nitrogen-doped carbon dots. We selected egg white as a cheap glycoprotein-based carbon source without the assistance of any chemicals. The synthetic process requires only 3 minutes during which carbonization and Nitrogen-doping are achieved at the same time. The fabricated carbon dots were characterized for particle size, structure and photoluminescence behaviour. The nanodots were amorphous, carbon rich, and naturally nitrogen doped particles with plentiful attached hydrophilic functional groups. They had average particle size of 2.98 ± 1.57 nm, emitted strong blue fluorescence and showed excitation-dependant emission behaviour. What is more, the practical use of this system for Raspberry Ketone determination in commercially available weight loss dietary supplement product was demonstrated successfully. In ethylene glycol medium, the addition of Raspberry Ketone enhanced the emission intensity of the synthesized carbon dots. The effects of reaction time and solvent were investigated. After optimization, the intensity enhancement was linear to the amount of Raspberry Ketone added to the assay solution in the concentration range 100-1000 ng/mL, with LOD and LOQ values of 15.10 and 45.45 ng/mL; respectively. The method was validated in accordance with ICH guidelines and further applied to assay of Raspberry Ketone capsules showing excellent results. Finally, green assessment was done using the Analytical Eco-scale, GAPI and AGREE metric tools and all proved the greenness of our proposal.
Section IV.C: Micellar-enhanced spectrofluorimetric assay of trans-Resveratrol using Triton X-100: Application to dietary supplement products’ regulation
A sensitive and simple synchronous spectrofluorimetric method was developed for the determination of trans-Resveratrol in its supplement product. The proposed method was based on enhancement of native fluorescence of the studied drug in the presence of non-ionic Triton X-100 micellar system. Several optimization experiments were conducted, including Δλ value, solvent, pH and different surfactants/macromolecules. In aqueous solution, the synchronous fluorescence intensity of trans-Resveratrol was increased by 10-fold in the presence of 0.05% w/v Triton-X 100 at Δλ = 80 nm. Relative fluorescence intensities at 369 and 375 nm were rectilinear over low and high concentration ranges 10-170 and 200-2000 ng/mL; respectively, with LOD and LOQ values of 1.78 and 5.38 ng/mL; respectively. Validation was done in accordance with ICH guidelines. The method was applied to the analysis of a commercial trans-Resveratrol product sold over the internet, showing excellent percent recoveries and statistically compared to another published HPLC method, showing insignificant statistical difference between both. Method greenness was also assessed using Analytical Eco-scale, GAPI and AGREE metric tools. The proposed micellar enhanced synchronous spectrofluorimetric method offers the advantages of greenness, simplicity, sensitivity, and good linearity of a wide concentration range, making it a good candidate for a wide range of applications.
Section IV.D: Multivariate calibration for dietary supplements’ regulation: Advanced chemometrics for the simultaneous spectrophotometric determination of 2-Phenethylamine, Caffeine, p-Hordenine and p-Synephrine
In this contribution, a comparison was highlighted between the chemometric models partial least squares (PLS-1), genetic algorithm-partial least squares (GA-PLS) and genetic algorithm-artificial neural network (GA-ANN) for the simultaneous spectrophotometric determination of 2-Phenethylamine, Caffeine, p-Hordenine and p-Synephrine in their dietary supplement formulation. A 4-Factor 5-level experimental design was used to establish a calibration set of 25 mixtures containing different ratios of the studied drugs. Validity of the proposed models was assessed using an independent validation set of 6 mixtures. Comparison showed that variable selection by genetic algorithm increased the prediction power of PLS-1 and ANN that GA-PLS and GA-ANN showed superiority over PLS-1 in solving the overlapped spectra of the mixture components. GA-PLS and GA-ANN were successfully applied to the analysis of supplement product containing the studied drugs. The two models were also statistically compared to another published HPLC and NMR methods, showing insignificant difference. Method greenness was confirmed using Analytical Eco-scale, GAPI and AGREE metric tools.
This thesis consists of 78 Figures, 59 Tables, 269 Refences and ends with an Arabic summary.