Thesis (Ph.D)-Cairo University, 2023.

Bibliography: pages 288-291.

This thesis entitled “Analytical Study on Some Anti-allergic Drugs” represents an analytical study
for quantitative analysis of some drugs treating allergic conjunctivitis namely; Alcaftadine (ALF) in its
single formulation, Antazoline (ANT) and Tetryzoline (TET) in their mixture form, and Antazoline
(ANT) and Xylometazoline (XLO) in their mixture form. The aim of work is to develop and validate
sensitive methods for analysis of the studied drugs in pure form, in presence of their degradation
products, pharmaceutical formulation and biological fluids; rabbit aqueous humor.
The thesis comprises three parts:
Part I: General Introduction and Literature Review
In this part, a brief idea about allergic conjunctivitis is presented. It also includes a general introduction
about chemical and physical properties of Alcaftadine, Antazoline, Tetryzoline, and Xylometazoline.
Finally, a literature review about different officials and reported analytical methods of the chosen
drugs in their raw form, pharmaceutical formulations, and biological fluids.
Part II: Published Papers
This part includes seven chapters
Chapter I: Novel Solid-Contact Ion-Selective Electrode Based on a Polyaniline Transducer Layer
for Determination of Alcaftadine in Biological Fluid
The current chapter constructs the fabrication of a novel ion selective electrode for determining ALF.
The Glassy carbon electrode (GCE) was utilized as a substrate in fabrication of electrochemical sensor
containing polyaniline (PANI) as an ion-to-electron transducer layer. PVC polymeric matrix and
nitrophenyl-octyl-ether were employed in designing the ion-sensing membrane (ISM). Potential
stability was improved, minimization of electrical signal drift was achieved and inhibition of water
layer formation at the electrode interface was attainted. It was achieved between the electronic
substrate and the ion-sensing membrane by PANI inclusion. The sensor dynamic linear range was
from 1.0 x 10-2
to 1.0 x 10-6 mol/L and 6.3 x 10-7 mol/L detection limit. Selectivity and capabilities of
the formed ALF sensor was tested in presence of its pharmaceutical formulation excipients as well as
its degradation products. Additionally, the sensor was capable of quantifying the studied drug in a
rabbit aqueous humor. Method’s greenness profile was evaluated by the means of Analytical
Greenness (AGREE) metric assessment tool.
Summary
VI
Chapter II: Spectrophotometric Platform Windows’ Exploitation for the Green Determination
of Alcaftadine in Presence of its Oxidative Degradation Product
This chapter presents the determination of ALF in its oxidative degradation product's presence by
applying comprehensive study comparative of four different green stability indicating
spectrophotometric approaches through successful exploitation of different spectrophotometric
platform windows. Window I; based on absorption spectrum zero order data manipulation using the
newly developed extended absorbance difference (EAD). Window II; based on derivative spectra by
second order derivative (D
2
) data manipulation. Window III; based on ratio spectra applying constant
multiplication (CM) and absorptivity centering via factorized ratio difference spectrum (ACT-FSRΔP)
methods data manipulation. Finally, window IV; based on derivative of ratio spectrum by virtue of
first derivative of ratio spectral (DD1
) method data manipulation. Calibration curves construction were
over linearity range; 1.0-14.0 µg/mL for ALF.
Chapter III: Impressive Merger Between Green Analytical Approaches and Quality- by- Design
for Alcaftadine Determination in Eye Drops and Rabbit Aqueous Humor, Application to
Stability Study by Two Validated Chromatographic Methods
This chapter represents two green chromatographic techniques, namely, HPLC and TLC to determine
ALF in pharmaceutical formulation, rabbit aqueous humor, and in presence of its degradate.
Developed HPLC technique in this work was succeeded to determine ALF in the presence of its
oxidative degradate via a fruitful integration between GAC and AQbD resulting in increasing
efficiency and minimizing environmental impacts. Utilizing an XBridge® C18 (4.6 × 250 mm, 5 µm)
column, optimized chromatographic separation was accomplished utilizing isocratic elution with an
ammonium acetate buffer (pH 5.0): ethanol (60:40, by volume) as the mobile phase, a flow rate of 1.5
mL/min, and UV detection at 282.0 nm. Over a concentration range of 0.1 to 8.0 µg/mL, linearity was
attained. Additionally, this work was succeeded to determine ALF through a stability indicating study
using thin layer chromatographic technique on TLC plates precoated with silica gel 60F-254 as a
stationary phase using a developing system consisting of dichloromethane: ethanol: ammonia (5:5:0.1,
by volume) and scanned at 282.0 nm with 0.5 - 20.0 µg/band linearity range.
Chapter IV: Novel Stability-Indicating TLC-Densitometric Method for Quantification of
Antazoline and Tetryzoline; Application to Pharmaceutical Formulation
This chapter discusses a thin-layer chromatographic method establishment to determine both ANT and
TET in both bulk drugs, formulation, and in presence of their degradation products with the
elucidation of degradate's structures. Upon using silica gel plates and by means of a developing system
consisting of ethyl acetate: ethanol: ammonia (8: 2: 0.1, by volume), the studied drugs separation was
achieved, and scanning was carried out at 220.0 nm for the separated bands with a 0.2 - 18.0 µg/band
concentration range for each of ANT and TET. Optimum separation was achieved with values Rf of
Summary
VII
0.42 and 0.69, for TET and ANT, respectively. Additionally, the studied drugs' stock solutions were
imperiled to different stress conditions. Both drugs were found to be vulnerable to alkaline hydrolysis
as well as oxidative degradation showing well resolved degradation products' peaks. While they held
out against acidic, thermal and photo degradation. Mass spectrometry was employed to elucidate the
degradates' structures.
Chapter V: Green TLC-Densitometric Method for Simultaneous Determination of Antazoline
and Tetryzoline: Application to Pharmaceutical Formulation and Rabbit Aqueous Humor
In this chapter, we developed an environmentally friendly thin-layer chromatographic method to
determine both ANT and TET in their pure forms, pharmaceutical formulation, and spiked aqueous
humor samples. Upon using silica gel plates and by means of a developing system consisting of ethyl
acetate: ethanol (5: 5, by volume), the studied drugs separation was achieved, and scanning was carried
out at 220.0 nm for the separated bands with a 0.2 - 18.0 µg/band concentration range for each of ANT
and TET.
Chapter VI: Exquisite Integration of Quality-by-Design and Green Analytical Approaches for
Simultaneous Determination of Xylometazoline and Antazoline in Eye Drops and Rabbit
Aqueous Humor, Application to Stability Study
This chapter implements a stability indicating HPLC method developed to simultaneously determine
XLO and ANT in their binary mixture, rabbit aqueous humor and cited drugs' degradates by applying
analytical quality-by-design (AQbD) combined with green analytical chemistry (GAC) experiment for
the first time. Optimal chromatographic separation was attained by means of an XBridge® C18 (4.6 ×
250 mm, 5 µm) column through isocratic elution using a mobile phase consisting of phosphate buffer
(pH 3.0): ethanol (60:40, by volume) at a 1.6 mL/min flow rate and 230.0 nm UV detection. Linearity
was acquired over concentration ranges of 1.0-100.0 µg/mL and 0.5-100.0 µg/mL for XLO and ANT,
respectively.
Chapter VII: Smart Spectrophotometric Methods for the Simultaneous Determination of
Antazoline and Tetryzoline using Zero-Order Regression Equations
This chapter represents a comparative study of smart green spectrophotometric methods for the
simultaneous determination ANT and TET in their binary mixture's existence without preliminary
separation steps. The proposed methods namely, factorized zero-order method (FZM), constant
multiplication coupled with spectrum subtraction (CM-SS) and normalized derivative subtraction
(DS). The methods showed good reproducibility and recovery with %RSD less than 2. Recoveries
were obtained via the regression equations constructed at the corresponding maxima for each of ANT
and TET at 248.0 nm and 219.0 nm, respectively. The specificity of the developed methods was
investigated by analyzing laboratory prepared mixtures and was successfully applied for the analysis
Summary
VIII
of pharmaceutical formulations containing the cited drugs. Furthermore, the usage of 0.1 N ethanolic
NaOH as a solvent gave us the advantage of applying greenness profile assessment which was
accomplished by means of four metric tools; namely: analytical greenness (AGREE), green analytical
procedure index (GAPI), analytical eco-scale, and national environmental method index (NEMI).
Part III: Submitted Papers
This part includes seven chapters
Chapter I: Novel Carbon Nanotubes/Gold Nanoparticles Modified Carbon Paste
Electrochemical Sensor for Antazoline Determination in Aqueous Humor
In this chapter, a novel electrochemical sensor based on carbon paste electrode (CPE) was developed
for determination of ANT in drug substance, different pharmaceutical formulations and in aqueous
humor. CPE was modified with nanoparticles multiwalled carbon nanotube (MWCNT) composite and
further decorated with gold nanoparticles using amperometric electrodeposition technique in order to
attain Au-NP@MWCNT/CPE for enhancing sensor sensitivity. Linearity was divided into two linear
segments and found to be 2×10-7
-2×10-6 (1
st Linear segment) and 2×10-6
-2×10-5
(2nd Linear segment)
with limit of detection and quantification of 1.77×10-7
and 5.37×10-7
, respectively.
Chapter II: Potentiometric Ion-selective Electrode for Determination of Antazoline in Different
Formulations and Biological Fluids using Biomimetic Receptors
In this chapter, an innovative ANT selective membrane sensor was developed to detect ANT in its pure
form, eye drop formulations, degradation products, and biological fluid. A sensor was fabricated using
ionophore calix[8]arene and compared with blank ionophore. Linear responses of ANT were obtained
utilizing sensors 1 and 2 in concentration ranges of 1.0×10-2
to 1.0×10-7 mole/L and 1.0×10-2
to 1.0×10-6
mole/L, respectively. Nernstian slopes of 58.486 and 51.2 mV/decade over pH 8.0 were observed using
sensors 1 and 2, respectively. The proposed approach was used to determine ANT in the presence of
degradation products and in biological fluid without the requirement for any pretreatment or separation
stages in both manufactured eye drops Trillerg®
sterile ophthalmic solution and Otrivine-Antistin®
Eye
Drops. Finally, the proposed method was assessed for its greenness profile using various greenness
assessment tools.
Chapter III: A Novel Eco-Friendly Stability Indicating UPLC Method for Determination of
Antazoline and Tetryzoline in Eye Drops and Rabbit Aqueous Humor
In this chapter, a sensitive and reproducible stability-indicating UPLC method was developed to
determine ANT and TET in pharmaceutical formulation, rabbit aqueous humor, and in presence of
their degradation products. An analytical column: Waters ACQUITY UPLC® BEH C18 (2.1 × 100
mm, 1.7) was utilized with isocratic elution by means of phosphate buffer (pH 2.0) accommodating
Summary
IX
0.1% tetrahydrofuran: ethanol (70: 30, v/v). The flow rate was 0.25 mL/min, and PDA detection was
done at 220.0 nm. Linearity was attained in the ranges of 0.5-100.0 µg/mL and 0.5-80.0 µg/mL for
ANT and TET, respectively. In addition, various stress conditions were imperiled to stock solutions of
the studied drugs. Drugs showed optimum resolved peaks of degradation products indicating their
vulnerability to alkaline hydrolysis and oxidative degradation. Degradates' structures were elucidated
by means of mass spectrometry.
Chapter IV: A Novel Green Spectrofluorimetric Method for Simultaneous Determination of
Antazoline and Tetryzoline in their Ophthalmic Formulation
A spectrofluorimetric method had been developed for determination of ANT and TET in their
pharmaceutical formulation. A combined application of synchronous spectrofluorimetry and second
derivative mathematical treatment was developed. The developed method depended on reacting the
cited drugs with Dansyl chloride being a suitable derivatizing agent generating highly fluorescent
derivatives measured at emission wavelengths; 703.0 and 642.0 nm after excitation wavelengths;
350.0 and 320.0 nm for ANT and TET, respectively. Synchronous spectrofluorimetry coupled with
second derivative mathematical tool was developed and optimized using fluorescence data manager
software generating second derivative peak amplitudes at 556.5 nm for ANT and 516.7 nm for TET.
Linear responses have been represented over a wide concentration ranges of 0.5-12.0 µg/mL and 0.5-
10.0 µg/mL for ANT and TET, respectively.
Chapter V: A Novel Spectrofluorimetric Determination of Antazoline and Xylometazoline in
their Ophthalmic Formulation; Green Approach and Evaluation
This chapter implements a synchronous spectrofluorimetry coupled with the second derivative
mathematical tool for ANT and XLO determination in their dosage form. The developed method
depended on reacting the cited drugs with dansyl chloride, a suitable derivatizing agent, to generate
highly fluorescent derivatives. The products formed were measured at emission wavelengths; 703.0
and 712.0 nm after being excited at wavelengths; 350.0 and 355.0 nm for ANT and XLO,
respectively. Synchronous spectrofluorimetry coupled with second derivative mathematical tool was
developed and optimized using fluorescence data manager software generating second derivative peak
amplitudes at 556.5 nm for ANT and 598.0 nm for XLO. Linear responses were obtained over a wide
concentration range of 0.5-12.0 µg/mL for ANT and 0.1-10.0 µg/mL for XLO.
Summary
X
Chapter VI: Novel Stability-Indicating TLC-Densitometric Method for Simultaneous
Determination of Antazoline and Xylometazoline with an Efficacious Exploitation of Green
Analytical Fundamentals: Application to Pharmaceutical Formulation and Rabbit Aqueous
Humor
This chapter implements thin-layer chromatographic method establishment to determine both ANT
and XLO in pure form, pharmaceutical ophthalmic formulation, and in spiked rabbit aqueous humor.
Adequate separation was acquired using silica gel 60 F254 TLC plates with a developing system
consisting of ethyl acetate: ethanol (5:5, by volume) and 230.0 nm scanning. A linear correlation was
obtained in ranges; 0.2 - 18.0 µg/band for ANT and 0.2 - 14.0 µg/band for XLO. In addition, various
stress conditions were imperiled to the stock solutions of the studied drugs. Drugs showed optimum
resolved peaks of degradation products indicating their vulnerability to alkaline hydrolysis and
oxidative degradation. Degradates' structures were elucidated by means of mass spectrometry.
Chapter VII: Impressive Application of Green Spectrophotometric Methods for the Unique Novel
Simultaneous Determination of Antazoline and Xylometazoline
This chapter represents green and sensitive six selective spectrophotometric methods developed for
ANT and XLO determination in pure form and pharmaceutical ophthalmic formulation. This
technique comprised multiple sequential processes performed using built-in spectrophotometer
software using zero, derivative, and/or ratio spectra of the investigated components. These techniques
included factorized zero-order method (FZM), ratio subtraction coupled with constant multiplication
(RS-CM), ratio difference (RD), derivative ratio (DD1
), factorized dual wavelength coupled with
spectrum subtraction (FDWL-SS), and second derivative coupled with spectrum subtraction (D
2
-SS).
These unique methods had a very strong capability for analyzing ANT and XLO challengeable
combinations.

تتناول هذه الرسالة دراسة تحليلية للتقدير الكمى لبعض المركبات الصيدلية التى تعالج الحساسية مثل الكافيتادين، أنتازولين، تيترزيولين، و زايلوميتازولين والهدف من هذه الرسالة هو أستخدام طرق تحليلية ذات درجة حساسية عالية بالاضافة إلى أختبار صلاحيتها لتحليل المركبات الصيدلية المنتقاة لهذه الدراسة فى المستحضرات الصيدلية و السوائل الحيوية.
و تحتوي هذه الرسالة على ثلاث أجزاء تتضمن التراث العلمي والدراسة العملية بالإضافة إلى المراجع وملخصين باللغتين العربية والإنجليزية.

مستخلص
تتناول هذه الرسالة دراسة تحليلية للتقدير الكمى لبعض المركبات الصيدلية التى تعالج الحساسية مثل الكافيتادين، أنتازولين، تيترزيولين، و زايلوميتازولين والهدف من هذه الرسالة هو أستخدام طرق تحليلية ذات درجة حساسية عالية بالاضافة إلى أختبار صلاحيتها لتحليل المركبات الصيدلية المنتقاة لهذه الدراسة فى المستحضرات الصيدلية و السوائل الحيوية. و تحتوي هذه الرسالة على ثلاث أجزاء تتضمن التراث العلمي والدراسة العملية. يلخص الجزء الأول بعض المعلومات حول المركبات الصيدلية التى تعالج الحساسية و مقدمة التراث العلمى لمادة الكافيتادين، أنتازولين، تيترزيولين، و زايلوميتازولين والطرق المنشورة لقياسهم في المادة الخام والمستحضرات الصيدلية و السوائل البيولوجية. و يشتمل الجزء الثانى الأبحاث المنشورة و يتضمن هذا الجزء سبعة فصول كما يشتمل الجزء الثالث الأبحاث المقدمة و يتضمن هذا الجزء سبعة فصول. الهدف من هذة الرسالة هو أستخدام طرق تحليلية ذات درجة حساسية عالية بالاضافة إلى أختبار صلاحيتها لتحليل المركبات الصيدلية المتنقاة لهذه الدراسة فى المستحضرات الصيدلية و السوائل الحيوية.

Issues also as CD.

Text in English and abstract in Arabic & English.