الفهرس 
Introduction to chronic Obstructive Pulmonary Disease (COPD)Only 14 pages are availabe for public view
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Abstract
The thesis consists of six parts:
Part I: Introduction to chronic Obstructive Pulmonary Disease (COPD)
This part includes a general introduction to chronic obstructive pulmonary disease (COPD); its definition, symptoms, risk factors and treatments including pharmacological and non-pharmacological ones.
Part II: Literature review of Investigated drugs
This part includes three sections:
Section A: Literature review of indacaterol
This section includes a review of the structure, physical properties, pharmacodynamics, pharmacokinetics and different reported methods for the analysis of indacaterol.
Section B: Literature review of glycopyrronium
This section includes a review of the structure, physical properties, pharmacodynamics, pharmacokinetics and different reported methods for the analysis of glycopyrronium.
Section C: Literature review of tiotropium
This section includes a review of the structure, physical properties, pharmacodynamics, pharmacokinetics and different reported methods for the analysis of tiotropium.
Part III: Spectroscopic techniques for determination of the investigated drugs
This part includes two sections:
Section A: Spectrophotometric methods for simultaneous determination of indacaterol and glycopyrronium based on signal processing techniques of ratio spectra
Three spectrophotometric methods have been developed and validated for simultaneous determination of indacaterol and glycopyrronium in their binary mixtures and novel pharmaceutical dosage form without the need for any separation processes. The proposed methods are based on different signal processing techniques of ratio spectra namely; Numerical Differentiation (ND), Savitsky-Golay (SG) and Fourier Transform (FT).
The developed methods are considered the first methods to be published for simultaneous determination of the two drugs.
The proposed methods showed good linearity over the concentration range of (1.00 – 30.00 and 10.00 – 35.00 μg/mL) for indacaterol and glycopyrronium, respectively. The LOD and LOQ calculated indicate the adequate sensitivity of the proposed methods.
The accuracy of pure forms of both drugs calculated as percentage recoveries were in the range of 99.00% – 100.49% demonstrating an excellent accuracy of the proposed methods. The low values of RSD% (< 1.5%) for inter and intra-day variation suggests an excellent precision.
The proposed methods were successfully applied for simultaneous determination of the IND and GLY in lab prepared mixtures, pharmaceutical dosage form and content uniformity test with excellent accuracy. The results obtained using standard addition technique indicates accuracy of the proposed methods for determination of IND and GLY in pharmaceutical formulation. There was no interference from other components in the matrix.
The results obtained by the proposed spectrophotometric methods were statistically compared with the reported methods and no significant difference was found.
Section B: Spectrofluorimetric determination of tiotropium using COOH functionalized CdTe quantum dots as fluorescence probe
TIO doesn’t possess native fluorescence. A new spectrofluorimetric method was developed and validated based on the quenching effect of tiotropium on functionalized COOH-capped quantum dots (CdTe QDs) as fluorescence probe. The fluorescence intensity was measured at λem 520 nm after excitation at λex 259 nm.
Other fluorophores reported in literature that can react with function groups of TIO need heating for long time resulting in TIO degradation or require further extraction step with organic solvents to the formed ion pair as which make the method tedious, time consuming and affect its accuracy and precision. The proposed method overcomes these disadvantages and provide rapid, simple, accurate and precise quantification for tiotropium.
Different factors affecting the method was optimized to obtain the maximum response. The studied factors were: buffer pH, type and volume, quantum dots concentration and volume and incubation time. The best quenching effect was reached using 1.50 mL of 0.10 M Tris-HCl buffer (pH 7.5) and 1.25 mL of 0.50 x 10-4 M QDs solution after incubation time 15 minutes.
The plot of Fo/F versus tiotropium concentration fit well with Stern-Volmer equation and showed good linearity over concentration range (1.00 – 50.00 µg/mL). The LOD and LOQ were found to be 0.32 and 0.97 (µg/mL) which indicates the adequate sensitivity of the proposed method.
The accuracy of determination of tiotropium in pure form calculated as percentage recoveries was in the range of 98.53% – 101.40% with low value of RSD% (1.189%) demonstrating an excellent accuracy of the method. The RSD% for repeatability (intra-day) and intermediate precision (inter-day) were found to be 0.961% and 1.501%, respectively. The low values of RSD% for intra and inter-day variation suggest an excellent precision of the method.
The method was successfully applied for determination of tiotropium in its pharmaceutical dosage form and content uniformity test with good accuracy.
The results obtained by the proposed spectrofluorimetric method were statistically compared with the official European pharmacopeia method and no significant difference was found.
Part IV: Electrochemical Techniques for Determination of Investigated Drugs
This part includes two sections:
Section A: Stable glycopyrronium bromide solid contact ion selective potentiometric sensors using multi-walled carbon nanotubes, polyaniline nanoparticles and polyaniline microparticles as ion-to-electron transducers; a comparative study
Glycopyrronium bromide solid contact ion selective potentiometric sensors using multi-walled carbon nanotubes (MWCNTs), polyaniline nanoparticles (PANI NPs) and microparticles (PANI MPs) as ion-to-electron transducers were fabricated and compared to transducer free blank sensor. Their inclusion in solid contact (SC) sensors offer shorter response time, lower potential drift and longer life time.
from a comparative point of view, inclusion of MWCNTs in the SC sensor offer longer lifetime (50 days) compared to PANI based SC sensors. While, the inclusion of PANI showed shorter response times. Hence, PANI-based sensors showed better stability on short term, while MWCNTs offered long term stability.
PANI NPs gave faster response than MPs. This may be attributed to the higher surface/volume ratio in NPs which enlarges the contact area with the polymeric membrane, increasing the transduction at the interface. Moreover, PANI NPs showed lower potential drift and relatively longer life time than MPs. Nano-sized particles result in smooth surfaces with a good surface coverage (protection) of the underlying SC.
The proposed sensors were successfully applied to content uniformity testing of GLY in a newly approved pharmaceutical dosage form with high accuracy.
The results obtained by the proposed sensors were statistically compared with a reported method and no significant difference was found.
Section B: Titanium (IV) Oxide Nanoparticles and Ionic Liquid Modified Carbon Paste Electrode for selective Voltammetric Determination of Indacaterol
No previous data were available in literature concerning the electrochemical behavior or determination of IND using voltammetry. Hence, the present work describes for the first time a new electrochemical sensing platform for voltammetric determination of IND. Moreover, the electrochemical redox mechanism of IND at the proposed sensor was studied.
Different factors were investigated to enhance the electrochemical response of carbon paste electrode (CPE) using cyclic voltammmetry. The first factor to be studied was the effect of different modifiers. the combination of titanium (IV) oxide nanoparticles (TiO2-NPs) and ionic liquid (IL) showed the best results due to excellent characteristics of TiO2-NPs and IL such as good electrical conductivity and high chemical stability and high surface area. The modification of CPE significantly enhances the peak current. The oxidation peak currents increased from 9.50 μA to 13.46 and 14.56 μA when TiO2-NPs and IL were used, respectively. However, the combination of TiO2-NPs and IL together showed the maximum current (17.14 μA) compared to using each modifier alone. Moreover, in case of modified carbon paste electrode (MCPEs), the potential shifted to less positive potential showing 493.27, 488.15 and 483.03 mV for TiO2-NPs-MCPE, IL-MCPE and TiO2-NPs-IL-MCPE, respectively, compared to 526.19 mV with CPE.
The electroactive surface area of the prepared electrodes was estimated by cyclic voltammetry using 1.00 x10-3 M K3Fe(CN)6 in 0.10 M KCl based on Randles- Sevcik equation and found to be 0.084, 0.113, 0.136 and 0.152 cm2 for CPE, TiO2-NPs-MCPE, IL-MCPE and TiO2-NPs-IL-MCPE, respectively. This confirms that the modification of CPE increased the surface area of the electrodes. The dramatic increase in oxidation current with the substantial negative shift of oxidation potential (decreasing the overpotential), indicates the enhancement of the electron transfer process and the catalytic ability of TiO2-NPs-IL-MCPE to IND oxidation.
Other factors as pH of the buffer solution, TiO2-NPs concentration and scan rate were also optimized. Measuring in Brittion-Robinson buffer (pH 7.0) using 3.0% (w/w) of TiO2-NPs at scan rate 100 mV s-1 were found to be the optimum conditions.
Other kinetics data of TiO2-NPs-IL-MCPE were calculated.
According to Laviron equation, the dynamic parameters of IND on TiO2-NPs-IL-MCPE were calculated found to be; Eo (0.4415 V), α (0.53) and kᵒ (2.838 s-1). The number of electrons transferred (n) = 2 which agrees with pH study.
from the chronoamperometric measurements of IND using TiO2-NPs-IL-MCPE, the diffusion coefficient was calculated based on Cottrell’s law and found to be 2.762 x 10-4 cm2 s-1.
The developed voltammetric method was validated according to ICH guidelines using square wave voltammetry. The novel TiO2-NPs-IL-MCPE demonstrated wide linear range (6.00 x 10-9 -2.00 x 10-5 M) in supporting electrolyte and (1.00 x 10-8 - 3.00 x 10-6 M) in spiked human urine. Moreover, The LOD and LOQ were found to be (1.30 x10-9 and 3.94 x10-9 M), and (2.50 x10-9 and 7.58 x 10-9 M) in supporting electrolyte and spiked human urine, respectively, which indicates the excellent sensitivity of the proposed sensor.
The accuracy calculated as mean percentage recoveries for IND in pure form was in 99.00% with low value of RSD% (1.324 %) demonstrating an excellent accuracy of the method. The RSD% for repeatability and intermediate precision were found to be 1.616% and 1.847%, respectively. The low values of RSD% for intra and inter-day variation suggest an excellent precision of the proposed sensor.
The proposed sensor enables the sensing of IND in the presence of the co-formulated drug GLY without any interference. The proposed sensor was shown to be successfully applied to the determination of IND in pharmaceutical samples, content uniformity test and human urine without any pretreatment steps.
The results obtained by the proposed sensors were statistically compared with a reported method and no significant difference was found.
Part V: Stability Studies on Tiotropium
This part includes three sections:
Section A: HPLC-SIAM for Determination of Tiotropium with LC-MS Structure Elucidation of Degradates
As no SIAM was reported in the literature for determination of TIO in presence of its degradates, a new HPLC-SIAM one was developed.
Buffer type and pH, organic modifier ratio, and detection wavelength were optimized to get the best separation.The mobile phase consisted of 20 mM ammonium acetate buffer adjusted to pH 5.8 with acetic acid (solution A) and acetonitrile (solution B) in ratio 70 : 30 (v/v), respectively. All analyses were carried at flow rate 1.0 mL/min and at room temperature using diode array detector at 240 nm.
Calibration curve was constructed in the range of (0.50 – 45.00 µg/ml). The LOD and LOQ were found to be 0.13 and 0.38 µg/mL which indicates the adequate sensitivity of the proposed method.
The accuracy calculated as percentage recoveries was in the range of 98.50% – 101.00% with low value of RSD% (0.961%) demonstrating an excellent accuracy of the method. The RSD% for repeatability (intra-day) and intermediate precision (inter-day) were found to be 1.111% and 1.158%, respectively which suggests an excellent precision of the method.
TIO peak in the acidic, alkaline and thermal degradation solution showed the same retention time of TIO standard (5.38 ± 0.03). Specificity of the method was confirmed by testing peak purity of TIO in presence of its degradation products in the mixture. The purity factor (999.979) was within the threshold limit (990.000) which indicates the purity of TIO peak and absence of coeluting compounds.
The measured system suitability testing (SST) parameters data of the proposed method for determination of TIO in presence of its degradates were found to be within the acceptance limits given by Ph. Eur.
The data showed that the measured SST parameters are still within the limits of acceptance after introducing small changes in the chromatographic conditions. Moreover, tR was almost unaffected and values of RSD% of the peak areas under the investigated changes did not exceed 2%.
The proposed method was successfully applied for determination of TIO in pharmaceutical dosage forms and in content uniformity test.
The results obtained by the proposed HPLC method were statistically compared with the official European pharmacopeia method and no significant difference was found.
The structure of the degradates was elucidated using LC-MS and the degradation pathway was suggested. The two suggested hydrolytic degradation products of TIO resulting from hydrolysis of the ester linkage under alkaline, acidic and thermal conditions are N-methyl scopine and 2, 2-dithienyl glycolic acid.
Section B: HPTLC-SIAM for Determination of Tiotropium
No stability assay indicating HPTLC method was found in literature for analysis of TIO. Accordingly, a new HPTLC-SIAM method for determination of TIO in presence of its degradates was developed and validated.
First different mobile phases were tried. However, none of the tried compositions showed good peak shape for both the drug and degradates. Finally, the mobile phase consisted of water: ethyl acetate: acetonitrile (1: 3: 5, v/v/v) showed the best results regarding peak shape and separation. Linear ascending development was carried out in the automatic developing chamber. The optimized chamber saturation time was 20 min at 25 ± 2 °C. The length of chromatogram run was 85 mm. After drying for 5 minutes, TLC plates were then scanned at 240 nm.
Calibration curve was constructed in the range of (40.00 – 500.00 ng/spot). The LOD and LOQ were found to be 17.48 and 38.18 ng/spot which indicates the adequate sensitivity of the proposed method.
The accuracy calculated as percentage recoveries was in the range of 98.65% – 101.50% with low value of RSD% (1.112%) demonstrating an excellent accuracy of the method. The RSD% for repeatability (intra-day) and intermediate precision (inter-day) were found to be 1.213% and 1.458%, respectively, which suggests an excellent precision of the method.
TIO peak in the acidic, alkaline and thermal degradation solution showed the same Rf of TIO standard (0.1 ± 0.02). The spot of TIO in the degradation solutions was well separated from the spot of the degradates (Rf = 0.35 ± 0.02) in different degradation mixtures. The peak purity of TIO spot isolated from the different degradation mixtures was assessed by comparing the spectra with TIO standard at different levels: peak start(s), peak maximum (m) and peak end (e) positions of the spot. Good correlation [r(s,m) 0.999973, r(m,e) 0.999673] was obtained between the standard and the sample overlain spectra. This confirms the specificity of the proposed method and absence of interference from the degradates in the mixture.
The measured SST parameters data of the proposed method for determination of TIO in presence of its degradates were found to be within the acceptance limits given by Ph. Eur. The measured SST parameters were still within the limits of acceptance after introducing small changes in the chromatographic conditions. Moreover, RSD% of the peak areas under the investigated changes did not exceed 2%. This ensures the excellent robustness of the proposed method.
The proposed method was successfully applied for determination of TIO in its dosage forms and in content uniformity test with excellent recoveries.
The results obtained by the proposed HPTLC method were statistically compared with the official European pharmacopeia method and no significant difference was found.
Section C: Kinetics Study on Tiotropium Alkaline, Acidic and Thermal Degradation
Kinetics studies for alkaline, acidic and thermal degradation of TIO have not been previously studied. The order of degradation reaction was found to be pseudo first order reaction. from the obtained result of this study, it can be concluded that, TIO degradation is temperature and pH dependent. By increasing temperature, the alkaline, acidic and thermal degradation rates have been increased while, t1/2 and t90 were decreased. Moreover, by increasing pH, the rate of degradation was also increased. Activation energies of TIO degradation in alkaline, acidic and neutral conditions were calculated from Arrhenius plots. Comparison of these results indicated that TIO is less stable in alkaline conditions than acidic conditions.
Part VI: General Discussion
This part includes comparisons between the different proposed methods for determination of the investigated drugs in this thesis. The comparisons includes the validation parameters, applications, advantages and disadvantages of each method.
This thesis is composed of 302 pages. It contains 61 tables and 82 figures and refers to 191 references.