development and validation of new colorimetric method for...
TRANSCRIPT
The National Ribat University
Faculty of Post Graduate Studies and Scientific Research
Development and Validation of New Colorimetric Method
for the Analysis of Propanolol Hydrochloride in Bulk and
Tablet Dosage Form
A thesis submitted in partial fulfillment for the requirement of the degree
of master in Drug Quality Control
By
Samar Ali Abd Alrazig Ali
B.Pharm (OIU) 2015
Supervisor:
Professor/ Elrasheed Ahmed Gadkariem
Professor of pharmaceutical chemistry
2018
II
ةالكريم اآلية
:قال تعاىل
اك أنت العلمي الحكمي ﴾ نامتنا ا لا ما عل
بحانك ل عل لنا ا ﴿ قالوا س
( 32سورة البقرة: اآلية )
III
Dedication
I am honored to dedicate this work to my mother for her
continuous support and encouragement.
IV
Acknowledgement
In the name of Allah the merciful, the most
compassionate all praise be to Allah, the lord of the
world, and prayers and peace be upon Mohamed his
servant and messenger.
First and foremost, I must acknowledge my limitless
thanks to Allah the ever-magnificent, the ever-thankful
for his help and bless. I owe a deep debt of gratitude to
my supervisor Prof. Elrasheed Ahmed Gadkariem who
worked hard with me from the beginning till the
completion of the present thesis; he has been always
generous during all phases of the thesis.
I would like to take this opportunity to say warm
thanks to my parents for their help, support and
encouragement.
Last but not least, deepest thanks go to all people who
took part in making this thesis real.
V
List of contents NO. Contents Page
Al-aya Alkarema II
Dedication III
English Acknowledgment IV
List of contents V
List of tables XI
List of figures XIII
List of Scheme XIV
English abstract XV
Arabic abstract XVIII
Chapter one
1.1. Introduction 1
1.1.1. Analytical chemistry 1
1.1.2. Quality control 1
1.1.3. UV spectroscopy 1
1.1.3.1. Beer's and Lamber's law 2
1.1.3.2. Stoichiometry of the reaction 2
1.1.3.2.1. Method of continuous variation 2
1.1.3.2.2. Molar-ratio method 3
1.1.3.3. Colorimetry 3
1.1.3.4. Oxidation-reduction reactions ( Redox reactions) 3
1.1.4. Analytical method development 4
1.1.5. Analytical method validation 4
1.1.6. Validation parameters 5
1.1.6.1. Linearity 5
1.1.6.2. Range 5
1.1.6.3. Precision 6
1.1.6.3.1. Repeatability 6
1.1.6.3.2. Intermediate precision 7
1.1.6.3.3. Reproducibility 7
1.1.6.4. Accuracy 7
VI
1.1.6.5. Limit of detection (LOD) 8
1.1.6.6. Limit of quantification (LOQ) 8
1.1.6.7. Sensitivity 9
1.1.6.8. Robustness 9
1.1.7. Method optimization 9
1.1.8. Propanolol hydrochloride (PPH) 9
1.1.8.1. Chemistry 9
1.1.8.2. Physicochemical properties 10
1.1.8.3. Pharmacology 10
1.1.8.4. Clinical uses 10
1.1.9. Ammonium cerium (IV) sulfate dihydrate 11
1.1.9.1. Chemistry 11
1.1.9.2. Physicochemical properties 11
1.1.10. Methyl orange (MO) 11
1.1.10.1. Chemistry 11
1.1.10.2. Physicochemical properties 12
1.2. Literature review 12
1.2.1. Titrimetric 13
1.2.2. UV-Visible Spectrophotometric methods 14
1.2.3 Other methods 19
1.3. Study objectives 20
1.3.1 General objective 20
1.3.2. Specific objectives 20
Chapter two
2 Materials and methods 21
2.1 Materials 21
2.2. Instruments and Equipments 22
2.3. Apparatus 23
2.4. Experiments 24
2.4.1. Preparation of reagents 24
2.4.1.1. Preparation of 5M hydrochloric acid 24
2.4.1.2. Preparation of 1M sulfuric acid 24
VII
2.4.1.3. Preparation of ceric ammonium sulfate solutions 24
2.4.1.3.1. Preparation of 450, 562, 675 and 900 μg/ml ceric
ammonium sulfate solutions
24
2.4.1.3.2. Preparation of 0.888×10-3 and 0.222×10-3 M ceric
ammonium sulfate
24
2.4.1.4. Preparation of methyl orange solutions 25
2.4.1.4.1. Preparation of 50µg/ml methyl orange solutions 25
2.4.1.4.2. Preparation of 0.888×10-3 and 0.222×10-3 M
methyl orange solutions
25
2.4.1.5. Preparation of standard and sample stock solutions 25
2.4.1.5.1. Standard stock solution 25
2.4.1.5.2. Preparation of 0.888×10-3 M propanolol
hydrochloride standard solution
25
2.4.1.5.3. Sample stock solution 26
2.4.2. Absorption spectra 26
2.4.2.1. Scanning for the maximum wave length 26
2.4.3. Optimization (one-factor-at -a time) 26
2.4.3.1. Shaking manually for 15min 26
2.4.3.2. Sonication time 27
2.4.3.3. Heating for different times at different heating
temperature (45, 70℃)
27
2.4.3.4. Acid volume 28
2.4.3.5. Shaker rate and time 28
2.4.3.6. Effect of cerric concentration 29
2.4.4. Validation 29
2.4.4.1. Linearity 29
2.4.4.2. Limit of detection 30
2.4.4.3. Limit of quantification 30
2.4.4.4. Accuracy 30
2.4.4.4.1. Spiked recovery method 30
2.4.4.5. Precision 31
2.4.4.5.1. Intra-day and Inter-day 31
VIII
2.4.4.6. Comparison between the developed and official
method
32
2.4.4.6.1. Single point assay 32
2.4.4.6.2. Official method 32
2.4.4.6.3. t-test for accuracy 33
2.4.4.6.4. F-test for precision 33
2.4.4.7. Robustness 33
2.4.4.8. Stability 33
2.4.8. Determination of the molar ratio between the
reactants
34
2.4.8.1. Determination of molar ratio between the ceric(IV)
and the drug using molar ratio method
34
2.4.8.2. Determination of the molar ratio between ceric
(IV) and methyl orange
34
2.4.8.2.1. Using job's method 34
2.4.8.2.2. Using molar ratio method 35
Chapter three
3 Results 36
3.1 development of the spectrophotometric method 36
3.1.1. Establishment of the maximum wave length 36
3.2. Method optimization (one factor at a time) 37
3.2.1. Shaking manually for 15min: 37
3.2.1.1. The effect of different manual shaking time
duration
37
3.2.1.1. Linearity for the standard 37
3.2.2. Sonication time 38
3.2.3. Effect of ceric concentration 39
3.2.4. Heating 39
3.2.4.1. Heating at 700 C for different time durations 39
3.2.4.2. Heating at 450 C for different time durations 40
3.2.5. Acid volume 40
3.2.6. Shaking time and rate 41
3.2.6.1. Effect of different shaking rate: 41
IX
3.2.6.2. Effect of different shaking time durations at rate
800 osc/min
41
3.3. Method Validation 42
3.3.1. Construction of the calibration curves 42
3.3.1.1. Standard calibration curve 42
3.3.1.2. Sample calibration curve 43
3.3.2. LOD and LOQ 44
3.3.2.1. Limit of detection (LOD) 44
3.3.2.1. Limit of quantification (LOQ) 44
3.3.3. Regression and validation parameter 44
3.3.5. Accuracy 45
3.3.5.1. Spiked recovery method 45
3.3.6. Precision 46
3.3.6.1. Intra-day 46
3.3.6.2. Inter-day 47
3.3.7. Comparison between the developed and the official
method
48
3.3.7.1. Single point assay 48
3.3.7.2. Official method 48
3.3.7.3. t-test for accuracy 49
3.3.7.4. F- test for precision 49
3.3.8. Robustness 50
3.3.9. Stability 50
3.3.10. Determination of the molar ratio between the
reactants
51
3.3.10.1. Determination of molar ratio between the ceric(IV)
and the drug using Molar Ratio method
51
3.3.10.2. Determination of the molar ratio between cerric
(IV) and methyl orange
53
3.3.10.2.1. Using Job's method 53
3.3.10.2.2. Using molar ratio method 55
Chapter four
4.1. Discussion 57
X
4.2. Conclusions 69
4.3. Recommendation 70
References
Appendices
XI
List of Tables
Table No. Table name Page No.
1 Materials used in the study. 21
2 Instruments equipments used in the study. 22
3 Glassware used in the study. 23
4 The effect of different manual shaking time
duration on the absorbance
37
5 The mean absorbance of seven calibration curves
when manual shaking for 15 min was used as a
catalyst
37
6 Serial dilutions for the mean standard calibration
curve (n= 3)
42
7 Serial dilutions for the mean sample calibration
curve (n = 3)
43
8 Regression and validation parameters from the
mean standard calibration curve
44
9 Recovery percent from standard addition method 45
10 Intra-day precision calculation data 46
11 Inter-day precision calculation data 47
12 The percentage of the sample by the developed
method
48
13 The percentage of the sample by the official
method
48
14 alculation of the mean and RSD for the developed
and the official methods.
49
15 The change in the absorbance in response to small
change in the wave length.
50
16 The stability of the color solution with time 50
17 The stoichometry between the ceric(IV) and the
drug using molar Ratio method
51
18 The stoichiometry between ceric (IV) and methyl
orange using Job's method.
53
XII
19 The stoichiometry between ceric (IV) and methyl
orange using molar ratio method
55
20 The absorbance of the standard when the sonicator
was used as a catalyst
78
21 The effect of different cerric concentration 78
22 The absorbance of the STD when heating at 700 C
was used as a catalyst
79
23 The absorbance of the STD when heating at 450 C
was used as a catalyst
79
24 The effect of different acid volume on the
absorbance while using the shaker for 15 min at
rate 400 osc/min.
80
25 Shows the effect of shaking by the shaker on the
absorbance at different rates
80
26 Time effect on the absorbance at shaking rate of
800 osc/min.
81
XIII
List of Figures
Figure No. Figure name Page No.
1 The chemical structure of PPH. 10
2 The chemical structure of methyl orange. 12
3 Scanning of the colored product versus the blank. 36
4 The mean calibration curve of the standard 38
5 The absorbance of the standard when the sonicator
was used as a catalyst
38
6 The effect of different ceric concentration when
the sonicator used for 15 min and 2ml of HCL (5
M) was used
39
7 The absorbance of the standard when heating at
700 C was used as a catalyst
39
8 Tthe absorbance of the standard when heating at
450 C used as a catalyst
40
9 The effect of different acid volume while using the
shaker for 15 min at rate 400 osc/min
40
10 Tthe effect of shaking by the shaker at different
rate
41
11 The effect of time durations when the solution was
shaken at rate 800 osc/min
41
12 Mean standard calibration curve ( mean
absorbance values, n = 3)
42
13 Sample calibration curve. 43
14 The plot of the molar ratio between ceric(IV) and
the drug using molar ratio method
52
15 The stoichiometry between ceric (IV) and methyl
orange using Job's method
54
16 Tthe stoichiometry between ceric (IV) and methyl
orange using molar ratio method
56
17 The difference in color manifested by the sample
and blank at ceric concentration of 900µg/ml
81
XIV
18 The difference in color between the sample and
blank when heating at 45℃ for 20, 25 and 30min
was used as a catalyst.
82
19 The differences in color between the sample and
blank when heating at 70℃ for 5 and 10 min was
used as a catalyst
82
20 The differences in color between the sample and
blank when heating at 70℃ for 5 and 15 min was
used as a catalyst
83
21 The shaking machine (Shaker) 83
22 The gradual increase in the color intensity in
response to increased sample concentration
84
List of Schemes
Scheme No. Scheme name Page No.
1 The probable oxidation reaction of lansoprazol 59
2 The reaction between the propanolol and the
excess Ce(IV)
60
3 The species formed on protonation of methyl
orangein acidic medium
60
4 The oxidation products of methyle orange 61
5 The reaction scheme for the formation of the
measured color
62
XV
Abstract
Background:
The pharmacological and therapeutic relevance of propanolol
hydrochloride (PPH) have lead to development of several methods for its
analysis. Colorimetric method, particularly which is based on cerium (IV)
sulfate spectrophtometry, has been established as being very sensitive for
PPH analysis.
Objective:
The objective of this study was to develop and validate a new
colorimetric method based on ceric (IV) sulfate for the analysis of PPH.
Method:
The proposed method is an indirect spectrophotometric one based on the
ability of cerium (IV) sulfate to oxidize PPH and to bleach the colour of
the indicator methyl orange. In this method, PPH reacted with a measured
excess of cerium (IV) sulphate in acidic medium and the unreacted
oxidant was determined by reacting with methyl orange (MO) followed
by absorbance measurement at 508 nm.
Results:
The optimized method obeys Beer's Lamber law over the concentration
range (0.5-3.5) μg/ml. The correlation coefficient (R2) was 0.99983 and
0.999 for the standard and sample calibration curve respectively. The
detection and quantitation limit were found to be (0.0899 and 0.2725)
μg/ml respectively. Moreover, the values of RSD% was found to be
smaller than 2 and that of (t) was found to be smaller than the tabulated
one (4.30). On the other hand the solution color was found to be stable for
more than two hours.
XVI
Conclusion:
A new applicable method for the determination of PPH has been
developed and validated as per the current ICH guidelines. The proposed
method is simple, rapid and based on the measurement of stable colored
species using ceric ammonium sulfate solution as a quantitative reagent.
The proposed method does not take more than 15–20 min and is among
the most sensitive reported for PPH. An additional advantage of the
method is that the absorbance is measured at longer wavelengths where
the interference from excipients is far less than at shorter wavelengths.
Recommendation:
It is recommended to conduct further optimization of the
spectrophotomeric technique that involve ceric (IV) sulfate as an
oxidizing reagent by utilizing experimental design.
Ceric (IV) sulfate must be further investigated with respect to its stability
and the effect of heat on the reactions in which it takes part as a reagent
so that optimization of the experimental conditions can be based on
concrete experimental evidence rather than relying on trial and error.
The method is recommended for routine use in pharmaceutical
laboratories as a part of industrial quality control.
XVII
المستخلص
:الخلفية
ه. طريقة قياس لتحليل طرق عدة طورت بروبانولولاللدواء والعالجية عقاقيريةبسبب االهمية ال
انها دقيقة (, وجدIVسيريوم) كبريتات التي تستند علي استخدام تلك خصوصا و الطيفي اللون
جدا في تحليل البروبانول.
االهداف:
التي تعتمد لتطوير و تأكيد فعالية طريقة جديدة من طرق قياس اللون الطيفي تهدف هذه الداسة
لتحليل البروبانول.( IVعلى كبريتات سيريوم )
:الطرق
سيريوم تكبريتا ان الطريقة المقترحة هي طريقة تحليل طيفي غير مباشرة مبنية على مقدرة
(IVعلى اكسدة البروبانول و )تم وزن عشرين حبة . يميثيل البرتقالال كاشف تخفيف لون
.درة ناعمةووطحنها لب
( في وسط IVتم تفاعل البروبانولول مع كمية فائضة من كبريتات سيريوم )في هذه الطريقة,
تمت مع الميثيل البرتقالي.المادة الؤكسدة بالتفاعل حمضي, بعد ذلك تم تحديد الكمية المتبقية من
نانومتر. 508قراءة االمتصاص في
:لنتائج
معامل ماكيروجرام. كان (3.5-0.5)تمتثل الطريقة لقانون بيير المبرت خالل نطاق تراكيز
الحد الكشفي أماعلى التوالي. 0.999و 0.99983االنحدار لكل من المادة القياسية والعينة هي
االنحراف المعياري وجد ان قيم مايكروجرام على التوالي. 0.2725و 0.0899كانا فوالكمي
ثابتا لون المحلول كما ظل (4.30وان قيمة )ت( اقل من القيمة المجدولة ) 2النسبي اقل من
الكثر من ساعتين.
:الخالصة
( الحالية. الطريقة ICHتم تطويروتقييم طريقة مفيدة لتحليل البروبانول بموجب تعليمات )
المقترحة سريعة, بسيطة, ومبنية على قياس جزيئات لونية ثابتة في محلول كبريتات سيريوم
XVIII
(IVالط .)دقة في دقيقة وهي من بين اكثر الطرق 15-10اكثر من ريقة المقترحة التستغرق
جي عالي تصاص يتم في طول موفائده اضافية وهي ان قياس االمب س البروبانولول وتمتازقيا
تدخل الشوائب اقل بكثير من الطول الموجي القصير. حيث يكون
التوصيات:
التي تتضمن استحدام قياس اللون الطيفي يوصى باجراء المزيد من تحقيق االمثلية لتقنية
( كعامل مؤكسد وذلك باستخدام التصميم التجريبي. IVكبريتات سيريوم )
( فيما يتعلق بثباتيته وتاثير الحرارة IVات سيريوم )كبريتيوصى باجراء مزيد من التقصي حول
علي الدليل اتحقيق االمثلية لظروف التفاعل مستندمما يجعل , يدخل فيها على التفاعالت التي
التجريبي الثابت بدال عن االعتماد على التجربة والخطأ.
كجزء من مراقبة جودة يوصى باستخدام الطريقة لالسخدام الروتيني في المختبرات الصيدالنية
الدواء الصناعية.
Chapter one
Introduction and literature review
1
Chapter one
1.1. Introduction:
1.1.1. Analytical chemistry:
Analytical chemistry is the science concerned with the chemical
characterization of materials both natural and artificial. It provides the
methods and tools needed for identifying the substances which may be
present in a material (qualitative) as well as exact amounts of the
identified substances (quantitative). [1]
Analytical chemistry plays an important role in quality control as the
quality of manufactured products often depends on measurement of the
constituents and proper chemical proportions. [1]
1.1.2. Quality control:
According to the Association of Official Analytical Chemists (AOAC)
quality control is a part of quality management focused on fulfilling
quality requirements. [2]
There are different methods for quality control including visible and
ultraviolet spectrometry which can be applied for quantitative analysis
using Bear's lambert law.
1.1.3. UV spectroscopy:
The principle on which UV spectroscopy depends is the manifestation of
the definite changes of the electronic structure of ions and molecules as a
result of absorption of electromagnetic radiation in the visible and
ultraviolet regions of the spectrum. [3]
2
1.1.3.1. Beer's Lambert's law:
Beer's Lambert's law is applied to determine the exact quantity of a
substance which absorbs light, and it states that the absorbance is
proportional to sample concentration and the path length.
Beer's Lambert’s law is represented by the following equation:
A = a b c Eq 1
Where A is the absorbance, a is constant, b pathlength, and c is the
analyte concentration.
In cases where more than one reagent is used, stoichiometric methods
can be applied to determine their molar ratio.
1.1.3.2. Stoichiometry of the reaction:
The stoichiometry of a metal-ligand complexation reaction can be
determined using one of two methods.[5]
1.1.3.2.1. Method of continuous variation:
Method of continuous variation ,also called job's method, is considered
the most popular. This approach is based on preparation of a series of
solutions such that the total moles of metal and ligand, ntotal, in each
solution is the same.
The data can be ploted as absorbance versus the mole fraction of metal or
the legand, and then the mole fraction of ligand or that of the metal at the
intersection point can be used to determine the mole number of the metal
and legend. After the extrapolation of the absorbance data, the relative
amount of ligand and metal in each solution is expressed as:[5]
3
ntotal = (nM)i + (nL)i Eq 2
(xL)i = (nL)i
ntotal Eq 3
(xM)i = 1 - (nL)i
ntotal = =
(nM)i
ntotal Eq 4
Y = nL
𝑛𝑀 =
XL
𝑋𝑀 =
XL
1− 𝑋𝐿 Eq 5
Where ntotal is the total moles of metal and legand, (nM)I and (nL)I are the
moles of metal and ligand in solution i respectively, (xL)i and (xM)i are
the mmole fractions for the ligand and mmetal respectively, y is the
number of moles of the legand MLy
1.1.3.2.2. Molar-ratio method:
The principle on which the method is based is that the amount of one
reactant, usually the moles of metal, is held constant while varying the
amount of the other reactant.
A plot of absorbance as a function of the ligand-to-metal mole ratio,
nL/nM, has two linear branches, which intersect at a mole–ratio
corresponding to the complex’s formula. [5]
1.1.3.3. Colorimetry:
Colorimetric analysis is based on the variation of the color of a system in
response to the change in concentration of some constituent.
The color is usually formed as a result of adding an appropriate reagent or
otherwise it may be inherent in the desired constituent itself.
Both colorimetry and spectrophotometry provide a simple means for
determining minute quantities of substances.[6]
1.1.3.4. Oxidation-reduction reactions ( Redox reactions):
Redox reactions are electron transfer reactions, in which some elements
experience an increase in their oxidation state (oxidized) while others
experience a decrease in their oxidation state (reduced).[7]
4
In case of organic compounds oxidation results in an increase in the
number of C – Z bonds (usually C – O bonds) or a decrease in the number
of C – H bonds. On the other hand reduction results in a decrease in the
number of C – Z bonds (usually C – O bonds) or an increase in the
number of C – H bonds.[4]
1.1.4. Analytical method development:
The main purpose of analytical methods during the process of drug
manufacture and development is to provide information about potency,
impurity, stability and bioavailability of the drug. Novel analytical
methods are developed to reduce the cost and time for better precision
and ruggedness. The demand for new analytical techniques in
pharmaceutical industries is constantly increasing due to the rapid
increase in pharmaceutical industries and constant production of drugs in
various parts of the world. In the development of a new analytical
procedure, the intended purpose and scope of the analytical method are
the two main factors influencing the choice of analytical instrumentation
and methodology.
Important parameters evaluated during the method development include
specificity, linearity, range, precision, accuracy, solution stability, limit of
detection, limit of quantification and robustness. These methods are
optimized and validated through trial experiments.[8]
1.1.5. Analytical method validation:
Statistical validation refers to the statistical treatment of the data
generated after accomplishing thorough investigation of various aspects
of possible determinate errors and applying the relevant corrections.
Effective statistical techniques are applied to render the fluctuating and
randomly scattered data into a better form that may be employed
intelligently. The graphs between absorbance and concentration yielded
through statistical treatment of the calibration data aided by
5
programmable calculators and micro-computers are fairly accurate and
more presentable than those produced manually.[4]
1.1.6. Validation parameters:
1.1.6.1. Linearity:
Linearity is a term used to indicate the liner response of the analytical
method which increases or decrease with analyte concentration. The
equation of the straight line represented as:
y= a + b x Eq 6
Where a is the intercept of the straight line with the y axis and b is the
slope of the line.[9]
A linear relationship should be evaluated across the range of the
analytical procedure. It may be demonstrated directly on the drug
substance (by dilution of a standard stock solution) and/or separate
weighings of synthetic mixtures of the drug product components, using
the proposed procedure. Linearity should be evaluated by visual
inspection of a plot of signals as a function of analyte concentration or
content. If there is a linear relationship, test results should be evaluated by
appropriate statistical methods. The correlation coefficient, y-intercept,
slope of the regression line and residual sum of squares should be
submitted. A plot of the data should be included. In addition, an analysis
of the deviation of the actual data points from the regression line may
also be helpful for evaluating linearity.[8]
1.1.6.2. Range:
The term range refers to the interval between the upper and the lower
concentration of an analye for which an acceptable level of precision and
accuracy has been established.[9]
6
1.1.6.3. Precision:
The spread of the results around a central value is referred to as precision
and may be expressed as the standard deviation or the variance.
According to the ICH guidelines precision may be considered at three
levels: repeatability, intermediate precision and reproducibility.
Good precision is indicated by the tendency of the data to cluster together
with an appropriately small (σ).
Standard deviation (SD) and relative standard deviation (RSD) are
expressed as: [9]
SD =√Σi(Xi− X) 2
n−1 Eq 7
RSD% = SD/ 𝑋 × 100 Eq 8
To asses precision in comparison to an official method or a reported
method, F value is interpreted.
Fcal = SDa
2
𝑆𝐷𝑏2 Eq 9
Where:
SDa2 = The larger SD
𝑆𝐷𝑏2= The smaller SD
1.1.6.3.1. Repeatability:
Repeatability is a measure of the closeness of agreement amongst a
cluster of experimental results obtained under the same operating
conditions over a short interval of time. The same analyst performs
7
experiments using the same equipment and batch of reagents within a
short time. Repeatability conditions allow the minimum amount of
variation in the replicated experiments.[9]
1.1.6.3.2. Intermediate precision:
Intermediate precision detects within- laboratory variation of precision.
When the analysis carried out by different analysts, on different days
using different equipment.[9]
1.1.6.3.3. Reproducibility:
Reproducibility expresses the precision between laboratories.[9]
1.1.6.4. Accuracy:
Accuracy is a measure of agreement between an experimental result and
its expected value. [7]
It is assessed by comparison of the method with a previously established
reference method such as a pharmacopoeial method. It can also be
expressed as percent recovery in relation to the known amount
of analyte added to the sample or as the difference between the known
amount and the amount determined by analysis.[9]
two sided t- test equation expressed as
tcal = X1 − X2
√SD12
n1 +
SD22
n2
Eq 10
Where:
tcal = the calculated t value.
X1 and X2= Mean percent values for the assay and the official method.
8
SD1 and SD2 = Standard deviations for the assay and the official method.
n1 and n2 = number of the samples for the assay and the official method.
% Recovery equation is expressed as
%Recovery = ( 𝐶𝑓− 𝐶𝑢 )
𝐶𝐴 × 100 Eq 11
Where Cf is the concentration of the fortified sample, Cu is the
concentration of the unfortified samples and CA is the concentration of the
analyte added to the test sample.
1.1.6.5. Limit of detection (LOD):
A statistical statement of the smallest analyte concentration that can be
reliably detected [9]. It is expressed as
LOD = 3.3.× residual standard diviatin
slope Eq 12
Sy/x =√Σi(yi−ӯi )2
n−2 Eq 13
Sy/x = Residual standard diviation.
1.1.6.6. Limit of quantification (LOQ):
The limit of quantification is defined as the smallest amount of analyte
which can be quantified reliably [9]. It is expressed as
LOQ = 10×residual standard deviation
slpoe Eq 14
9
1.1.6.7. Sensitivity:
Sensitivity indicates the ability of the method to respond to a small
change in the analyte concentration.[9]
1.1.6.8. Robustness:
Robustness of an analytical procedure is a measure of how resistant the
precision and accuracy of an assay to small but deliberate variations and
provides an indication of its suitability during normal usage.
1.1.7. Method optimization:
Optimization methods are designed to provide the best values of system
design and operating variables. Optimization procedures should yield
values that will lead to the highest levels of system performance.[10]
One-factor-at-a time (OFAT) experiments are often performed which
[11]ng others fixed.vary only one factor or variable at a time while keepi
1.1.8. Propanolol Hydrochloride (PPH):
1.1.8.1. Chemistry:
Propranolol hydrochloride (PPH) is chemically known as (RS)-1-
isopropyl amino-3-(1-naphthyloxy) propan-2-ol hydrochloride (Figure 1).
It has the molecular formula C16 H21 NO2 .HCl and its molecular weight
is 295.81.PPH has the following structure:[12][13][14]
10
Figure 1: Illustrates the chemical structure of PPH.
1.1.8.2. Physicochemical properties:
Propanolol Hydrochloride is a white or almost white powder readily
soluble in water and in 95% ethanol, slightly soluble in chloroform and
insoluble in non polar solvents.[14]
1.1.8.3. Pharmacology:
Propranolol hydrochloride is a non-selective competitive antagonist of
endogenous and exogenous sympathomimetic amines at beta-adrenergic
receptors (Beta1 and Beta2).[12][13][14]
1.1.8.4. Clinical uses:
PPH as a beta-blocker is commonly used in the management of
hypertension, angina pectoris, cardiac arrhythmia, hypertrofic obstructive
cardiomyopathy, myocardial infarction, anxiety, essential tremor and
migraine. It is considered the most effective beta-blocker for prevention
of migraine. It may work by stabilizing arteries or preventing the central
generator of migraine in the brainstem from firing.[14]
11
1.1.9. Ammonium cerium (IV) sulfate dehydrate:
1.1.9.1. Chemistry:
It is also known as ceric ammonium sulfate dihydrate and ammonium
ceric sulfate dihydrate. Its molecular formula (NH4)4Ce(SO4)4.2H2O , and
its molecular weight is 632.55.[15]
1.1.9.2. Physicochemical properties:
Ammonium cerium (IV) sulfate dihydrate exists in the form of yellow to
orange crystals or powder.[14]It is a very strong one-electron oxidant in
acidic medium. The nature and concentration of the acid greatly influence
the redox potential of Ce4+/Ce3+ coupled.
The redox potential is + 1.28 V in 1M HCL, + 1.44 V in 1 M H2SO4, +
1.61 V in 1 M HNO3, + 1.7 V in 1 M HCLO4, and it is as high as + 1.87
V in 8 M HCLO4.
The redox potential thus increases in the order: hydrochloric acid <
sulfuric acid < nitric acid < perchloric acid. Whereas the redox potential
increases with increasing concentrations of perchloric acid, it decreases
with increasing concentrations of nitric acid and sulfuric acid.[16]
1.1.10. Methyl orange (MO):
1.1.10.1. Chemistry:
Methyl orange(CI.13025); Acid Orange 52; [p-(Dimethylamino)-
Phenylazo]benzene sulfonic acid, sodium salt; C14H14N3O3SNa.
FW=327.3g/mol[17] (Figure 2).
12
Figure 2: Illustrates the chemical structure of methyl orange.
1.1.10.2. Physicochemical properties:
Methyl orange characterized by its clear and distinct color change is
frequently used in titrations as a pH indicator (at pH 3.0(pink)-
pH4.4(yellow)) .[17]
The wavelength of maximum absorption of the MO sample can be 320 or
465 or 510 nm depending on the medium pH.[18]
1.2. Literature review:
For the assay of PPH in pharmaceutical formulations a number of
methods based on techniques such as titrimetry, UV/visible
spectrophotometry and chromatography have been developed.
Both BP [19] and USP [20] describe UV spectrophotmetric methods for the
assay of PPH after extraction into methanol.A potentiometrric method
involving the titration of the drug in ethanol against 0.1 M NaOH is also
described in IP. Infrared Absorption Spectroscopy for determination of
PPH is also reprted in IP.[21]
A brief review of these methods is presented in detail in the following
paragraphs.
13
1.2.1. Titrimetric:
Two simple and rapid titrimetric methods were developed by Iona and
Alexandra (2017). They were conducted in heterogeneous system (water /
chloroform) for the assay of clemastine fumarate and propranolol
hyhrocloride by ion association titration are proposed. Sodium lauryl
sulfate was used as a titration reagent. The titrations were carried out in
acidic medium, in the presence of sulfuric acid. The indicator for
titrations was dimethyl yellow. The reactions stoichiometry was found to
be 1:1 for both drugs. These methods were successfully applied to the
assay of clemastine fumarate and propranolol hydrochloride in bulk and
pharmaceuticals, with good accuracy and precision and without
detectable interference by excipients. Results are in good agreement with
those of pharmacopoeial methods (mean recovery was 99.89% and the
RSD% was 0.01% and 0.09% for repeatability and intermediate precision
respectively). [22]
An accurate and precise titrimetric method for the determination of PPH
with cerium (IV) sulphate was developed by Kanakapura et al (2003) the
method is based on the oxidation of the drug by a known excess amount
of cerium(IV) sulphate and back titration of the unconsumed oxidant with
ammonium ferrous sulphate. The procedures described were successfully
applied to the determination of propranolol hydrochloride in bulk drug
form and in tablets (range of error for accuracy was less than 2.5% the
RSD% was less than 2.5% for both repeatability and intermediate
precision).[23]
Potassiumdipertelluratocuprate (III) as an oxidizing reagent was used in a
titrimetric method for the determination of PPH. The sample was first
oxidized with the reagent in acidic media at room temperature,then
known volume of 10%KI was added and the liberated iodine was titrated
with 0.01N sodium thiosulphate using starch as indicator.[21]
14
A direct titrimetric method based on the bromination-oxidation reaction
was reported by Basavaiah et al (2003)[24] . It involves generation of
bromine in situ by the action of acid on bromate-bromide reagent.
Applicability of the method is over a concentration range of 3.0-18.0 mg
of PPH.
The oxidation of PPH by chloramine-T in acid medium was exploited to
develop a direct titrimetric method using methyl orange indicator [25] over
a concentration range 3.0-10.0 mg.
Basavaiah et al (2003) [26] reported another method based on the oxidation
of the drug by a known excess amount of sodium metavanadate in acid
medium and back titration of the unconsumed oxidant with ammonium
ferrous sulphate using N-phenyl anthranilic acid indicator. The method is
applicable over a concentration range 3.0-10.0 mg.
Basavaiah et al (2003)[27] also reported a method in which the drug
solution (1.0-9.0 mg) is treated with a known excess of silver nitrate and
after the precipitation is complete, unreacted silver nitrate is back titrated
with potassium thiocyanate using iron(III) alum as indicator.
Electro-analytical titrations like conductometric titration [28] were also
reported for the quantification of PPH in pharmaceuticals.
1.2.2. UV-Visible Spectrophotometric methods:
The determination of PPH by UV-spectrophotometric methods is
frequently reported in literature. Methods involve reactions with
hydroxyl, secondary amine group or chloride content of hydrochloric salt
of drug.
Kudige and Kanakapura[13]developed and validated simple, selective and
economic spectrophotometric methods for the analysis of the propranolol
hydrochloride (PPH) in bulk drug and pharmaceuticals. The methods
were based based on the oxidation of PPH with cerium(IV) in acid
medium. Excess cerium(IV) was measured by its reaction with p-
15
dimethylamino benzaldehyde (p- DMAB) and measuring the resulting
color at 460 nm (method A) or by its reaction with sulphanilic acid
(SAA) to give a pink colored product peaking at 540 nm (method B).
Linear relationships with good correlation coefficients were found in the
concentration ranges of 0.4–7.0 μg ml-1 for method A and 10–200 μg ml-
1for method B, respectively.
Rashanth and Basavaiah (2012)[29] described two simple and selective
spectrophotometric methods for the determination of propranolol
hydrochloride (PPH) as base form (PPL) in bulk drug, and in tablets and
capsules. The methods were based on the molecular charge-transfer
complexation of propranolol base (PPL) with either 2,4,6-trinitrophenol
(picric acid; PA) or 2,4-dinitrophenol (DNP). The yellow colored radical
anions formed on dissociation, were quantitated at 425 nm (PA method)
or 415 nm (DNP method). The assay conditions were optimized. Beer's
law was obeyed in the concentration ranges 2.4-42.0 μg ml-1 in PA
method and 9.0-126.0 μg ml- 1in DNP method.
Ahad et al (2017)[30] developed a simple, rapid and sensitive
spectrofluorimetric method for the determination of propanolol
hydrochloride in pharmaceutical formulations. The method was based on
the oxidation of the drug with Ce (IV) to produce Ce (III), and its
fluorescence was monitored at 356 ± 3 nm after excitation at 254 ± 3 nm.
Kanakapura. et al (2003)[23] developed a spectrophotometric method
based on the oxidation of the drug by a known excess amount of
cerium(IV) sulphate. This was followed by treating the unreated
cerium(IV) sulphate with iron(II) sulphate. The iron(III) sulphate
produced was complexed with thiocyanate and measured at 480 nm,
thereby permitting the determination of the amount of unreacted
cerium(IV) sulphate. Beer's law was obeyed for 0-5 µg mL-1 of
propranolol hydrochloride. The procedures described were successfully
16
applied to the determination of propranolol hydrochloride in bulk drug
form and in tablets.
Basavaiah et al (2003)[28] proposed a spectrophotometric procedure
involving the reaction of the drug chloride with mercury (II) thiocyanate
to form soluble mercury (II) chloride with the liberation of thiocyanate
ions. The reaction of the liberated thiocyanate ions with iron (III)
resulted in the formation of the familiar red color which was measured at
460 nm.
Basavaiah et al (2003) [26] also reported a method in which the unreacted
cerium(IV) sulphate was treated with iron(II) and the resulting iron(III)
was complexed with thiocyanate and measured at 480 nm in the
Beer’s law range 1.0-5.0 μg mL−1.
Basavaiah et al (2014) [25] also reported a similar method based on the
oxidation of PPH by a known excess of chloramine-T in acid medium.
This was followed by determination of the unreacted oxidant by reacting
with metol and sulphanilic acid at 520 nm. This method is linear over the
concentration range of 1.5-15.0 μg mL−1.
Another spectrophotometric method reported by Basavaiah et al
(2003)[27] involved the determination of the unreacted oxidant
metavanadate by reacting with diphenylamine. Absorbance was measured
at 560 nm in the concentration range 0.4-4.0 μg mL−1.
Basavaiah et al (2003) [24] also reported a method based on the addition of
a known excess of bromate-bromide mixture to an acidified solution of
the drug. The unreacted bromine was then determined by its bleaching
action on methyl orange color followed by measuring absorbance at 510
nm over the linear range 0.5-3.5 μg mL−1.
El-Didamony (2010)[31] developed three methods based on oxidation-
bromination reaction of PPH. Bromine was generated in situ by the action
17
of acid on a bromate-bromide mixture, followed by determination of
unreacted bromine by three different reaction schemes. The residual
Bromine in the first method was determined by indigo carmine dye
and absorbance was measured at 610 nm over the linear range 1.0-13.0
μg mL−1. The other two methods involved determination of the residual
bromine by treating a known excess of iron(II) a complexation of the
resulting iron (III) with thiocynate or the residual iron(II) with 1,10-
phenanthroline. Absorbance was measured at 480 and 510 nm,
respectively with corresponding concentration ranges 4.0-12.0 μg mL−1
and 2.0-9.0 μg mL−1.
Two procedures, similar to the above, were reported by Gowda et al
(2002)[32] based on oxidizing PPH by a known excess of N-
bromosuccinimide (NBS) in H2SO4 medium. This was followed by the
reaction of the unreacted oxidant with promethazine hydrochloride (PH)
or methdilazine hydrochloride (MDH) to yield red colored products.
Maximum absorption was encountered at 515 and 513 nm, respectively
and the corresponding Beer’s law ranges were 0.5-12.5 μg mL−1 and 0.3-
16.0 μg mL−1.
Al-Attas et al (2006)[33] described a method based on oxidizing PPH by a
known excess of NBS, in an acidic medium. The excess oxidant was then
reacted with amaranth dye and the absorbance measured at 521 nm over
the concentration range 2.0-6.4 μg mL−1.
A new spectrophotometric method developed by Sharma and jasvir
(2018) [12] was based on the derivatization of the amino function present
in propanolol hydrochloride to the corresponding yellow copper (I) drug
dithiocarbamate derivative through reaction with carbon disulphide,
pyridine and copper (I) perchlorate in aqueous acetonitrile. Absorbance
was measured at 406 nm for propranolol and 400 nm for metoprolol.
18
In addition to the indirect methods mentioned above, several direct
methods based on a variety of reaction chemistries are also encountered
in literature.
Amod et al (2011)[34] developed two rapid and economical Q-Absorbance
and Multicomponent UV Spectrophotometric Methods for Simultaneous
Estimation of Propranolol Hydrochloride and Flunarizine
Dihydrochloride in Capsules. Both methods were successfully applied for
the routine analysis of PRH and FNZ in bulk and combined capsule
dosage form.
Naulay et al (2015)[35] developed and validated a method involving
Simultaneous estimation of Hydrochlorothiazide and Propranolol in
dosage form by Simultaneous Equation Method. The wavelengths
selected for the method were at the λmax 271nm and 289nm for
Hydrochlorothiazide and Propranolol respectively. The linearity of
Hydrochlorothiazide and Propranolol was found to be in the range of 4-
24 μg/ml & 8-48μg/ml respectively.
Idowu et al (2004)[36] reported a method in which diazotized 4-amino-3,5-
dinitrobenzoic acid (ADBA) coupled with drug and the resulting azo-dye
was measured at 490 nm in the concentration range of 1.0-8.0 μg mL-1.
A method reported by Bhandari et al (2008)[37] involved the reaction of
PPH with 1-chloro-2,4-dinitrobenzene, forming a complex, which absorb
maximally at 314.6 nm.
Two methods were reported by Golcu et al (2004)[38], the first method
was based on treating PPH with copper(II) and the formed colored
complex measured at 548 nm in the concentration range 2x10-5- 1×10-2
whereas the second method was based on treating PPH with cobalt(II)
and the formed complex measured at 614 nm over the same linear range
as the first method.
19
El-Ries et al (2000) [39] reported two spectrophotometric methods based
on the
charge-transfer complex reaction of PPL with π-acceptors,
tetracyanoethylene, or chloranilic acid to give highly coloured complex
species which is quantitated spectrophotometrically at 415 or 510 nm
with corresponding linear ranges of 10.0- 160.0 and 2.0-25.0 μg mL-1.
Similar reactions used by Salem [40] were based on the reaction of PPL as
n-electron donor with the sigma-acceptor iodine and π-acceptors such as
7,7,8,8-tetracyaniquinodimethane, 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone, tetracyano ethylene, bromanil and chloranil. Measuring of
the resulting C-T complexes was done at 365, 840, 420, 470, 450 and 440
nm respectively and Beer’s plots were obeyed in a general concentration
range of 4.0–120.0 μg mL−1 for all the methods.
A method reported by Hussain et al [41] involved the redox reaction of
PPH with cerium(IV) in H2SO4 medium on heating and the developed
color was measured at 478 nm.
El-Emam et al [42] developed a method based on oxidative-coupling
reaction in which a mixture of an acidic solution of MBTH and PPH was
treated with cerium(IV) and the resulting orange color peaking at 496 nm
was measured and the method is applicable over the linear range of 1.0-
10.0 μg mL−1.
1.2.3 Other methods:
Literature also cites several HPLC procedures designed for the
separation and determination of components in binary combinations
containing PPH
along with some other drugs, such as flunarizine dihydrochloride[43] ,
Diazepam[44], Alprazolam[45], and furosemide. [46]
Other chromatographic techniques like gas chromatography [47]are also
found in the literature.
20
Other techniques reported for the assay of PPH include atomic absorption
spectrometry [48], condutmetry [49], and voltammetry. [50]
Visible spectrophotometry compared to chromatographic and other
techniques reported till date, is the most frequently used and is based on
different reaction schemes.
However, most of the reported visible spectrometric methods are face
with one or another limitation such as poor sensitivity, narrow linear
range, scrupulous control of experimental variables, longer contact times,
extraction step or heating step and use of organic/expensive chemicals.
1.3. Study objectives:
1.3.1 General objective:
To develop a simple, accurate and precise colorimetric method for the
analysis of propanolol hydrochloride in bulk and tablet dosage form.
1.3.2. Specific objectives:
1. To optimize the conditions used in the method.
2. To investigate the applicability of Job's method and Molar ratio
method for the determination of the molar ratio of the reactant.
3. To validate the developed method according to ICH guidelines.
4. To apply the method for the analysis of propanlol tablets.
Chapter two
Materials and Method
21
2. Materials and methods:
2.1 Materials:
Table 1: Illustrates materials used in the study.
Materials Batch No. Mfg
date
Exp.
date
Company country
Propanolol
HCL
standard
M170213 14-2-
2017
5-11-
2018
CIMA Sudan
Propanolol
HCL
tablets
TB/058/18865 - - CIMA Sudan
Ceric
ammonium
sulfate
OX010929 - - QualiKems India
Methyl
orange
12380901 2-2016 - Sharlua Spain
H2SO4 16153811 - 2-2020 Sharlua Spain
HCL 21.0020212.2000 - 12-
2018
CHEM-
LAB
Belgium
Methanol G16A/0316/2906/13 7-2016 6-2021 SDFCL India
22
2.2. Instruments and Equipments:
Table 2: Illustrates instruments and equipments used in the study.
Apparatus Model/serial No. Company Country
UV/VIS
Spectrophotometer
[single beam]
6305 JENWAY UK
Sonicator
[Ultra sonic bath]
301.0052748.038 BANDELIN
SONOREX
Germany
Electronic balance BAS32 BOECO Germany
Heating mantile 1g404 BTI India
Flask shaker R000102248 Stuart UK
Thermometer - RAMIN
DELUXE
-
23
2.3. Apparatus:
Table 3: Illustrates glassware used in the study.
Equipment Size Company Country
Beaker 50,100,200,250 Boro 3.3 -
Measuring
cylinder
10,25,100 Hirschamann
Germany
Nylon
syringe filter
0.45µg Olimpeak -
Glass funnel ISOLAB Germany
Graduated
pipette
1,5 ISOLAB Germany
Micro
pipette
1 ISOLAB Germany
Mortar and
pestle
- - -
Volumetric
flask
10,25,50,100,200,500 ISOLAB Germany
Volumetric
pipette
1,2,5,10 ISOLAB Germany
24
2.4. Experiments:
2.4.1. Preparation of reagents:
2.4.1.1. Preparation of 5M hydrochloric acid:
A volume equivalent to 208 ml of 37% HCL was transferred into a 500
ml volumetric flask and diluted to the mark with distilled water.
2.4.1.2. Preparation of 1M sulfuric acid:
A volume equivalent to 28 ml of 98.7% sulfuric acid was transferred into
a 500 ml volumetric flask and the volume was made up to the mark with
distilled water.
2.4.1.3. Preparation of ceric ammonium sulfate solutions:
2.4.1.3.1. Preparation of 450, 562, 675 and 900 μg/ml ceric
ammonium sulfate solutions:
Weights of ceric (0.0225, 0.0281, 0.0337, and 0.045) g were transferred
separately into a series of 50 ml volumetric flasks. The contents of the
volumetric flasks where then made up to 50 ml using 1 M sulfuric acid
for the preparation of different ceric concentrations (450, 562, 675 and
900) μg/ml.
2.4.1.3.2. Preparation of 0.888×10-3 and 0.222×10-3 M ceric
ammonium sulfate:
The same procedure mentioned above for the preparation of 562 μg/ml
was followed to prepare (0.888×10-3) M of ceric ammonium sulfate. The
resultant solution was used to prepare (0.222×10-3) M by taking 12.5 ml
of it and transferred into a 50 ml volumetric flask. The volume was
completed to the mark with 1 M sulfuric acid.
25
2.4.1.4. Preparation of methyl orange solutions:
2.4.1.4.1. Preparation of 50µg/ml methyl orange solutions:
For the preparation of 50µg/ml of methyl orange solution, an accurately
weight amount of methyl orange (0.01 g) was transferred into a 200 ml
volumetric flask. The volume was completed to the mark with distilled
water.
2.4.1.4.2. Preparation of 0.888×10-3 and 0.222×10-3 M methyl orange
solutions:
To prepare (0.888×10-3) M of methyl orange solution, 0.0290g was
weighed and transferred to 100-ml volumetric flask and the volume was
completed to the mark by distilled water.
The resultant solution was used to prepare (0.222×10-3) M by taking 12.5
ml of it and transferred into a 50 ml volumetric flask. The volume was
completed to the mark with distilled water.
2.4.1.5. Preparation of standard and sample stock solutions:
2.4.1.5.1. Standard stock solution:
A weight equivalent to 10 mg of the standard was placed into a 50 ml
volumetric flask and the volume was made up to the mark with distilled
water. 5 ml from the resultant solution was then further diluted to 100 ml
volumetric flask and diluted to the mark with distilled water (solution A).
2.4.1.5.2. Preparation of 0.888×10-3 M propanolol hydrochloride
standard solution:
0.0230 g of PPH was accurately weighed and transferred into a 100 ml
volumetric flask and the volume was made up to the mark with distilled
water (solution B).
26
2.4.1.5.3. Sample stock solution:
Twenty tablets were weighed and ground into a fine powder using a
mortor and pestle. A weight equivalent to 10 mg PPH of the ground
tablets was placed in a 50 ml volumetric flask and the volume was made
up to the mark with distilled water. 5 ml of the resultant solution was
transferred into a 100 ml volumetric flask and diluted to the mark with
distilled water (solution C).
2.4.2. Absorption spectra:
2.4.2.1. Scanning for the maximum wave length:
For preparing a concentration of 2 µg/ml of standard solution, 2 ml of the
solution A was transferred into a 10 ml volumetric flask. 1 ml of 5 M
HCL, 3 ml distilled water and 1 ml of 562 µg/ml ceric ammonium sulfate
solution were then added to the flask. The shaker was used for shaking
the contents of the volumetric flask at a rate of 300 osc/min for 15
minutes. This was followed by adding 1 ml of 50µg/ml methyl orange to
the mixture in the flask and completing the volume to 10 ml with distilled
water. A blank was prepared following the same procedure above with
the exception of using the standard. The solution was scanned by UV
visible spectrophotometer in the range of 200-700 nm and the maximum
wavelength was determined.
2.4.3. Optimization (one-factor-at -a time):
2.4.3.1. Shaking manually for 15min:
Serial dilutions of the standard stock solution were prepared by micro
pipetting (0.5, 1, 1.5, 2, 2.5, 3) ml into six 10 ml volumetric flasks. To
each of these flasks 1 ml HCL (5 M) and 1 ml ceric ammonium sulfate
solution (562µg/ml) and 3 ml distilled water were added. The flasks were
27
then exposed to manual shaking for 15 min followed by adding 1 ml
methyl orange (50µg/ml) to each flask and making the volume up to the
mark with distilled water. A blank was prepared under the same
conditions omitting the addition of the standard. Linearity was then
scanned several times.
2.4.3.2. Sonication time:
For testing sonication time, 2 ml of the standard stock solution was
transferred into set of eight 10 ml volumetric flasks. To each of the flasks
1 ml of 562 µg/ml ceric ammonium sulfate, 1 ml of 5 M HCLand 3 ml
distilled water were added. The flasks were then sonicated for
(5,10,15,20,25,30,35,40) min, after shaking time completed, 1 ml methyl
orange (50µg/ml) was then added to each flask and the volume made up
to the mark with distilled water. A blank was prepared for each of the 8
samples under the same conditions omitting the addition of the standard.
Two 100 ml beakers were filled with about 40 ml water and placed in the
sonicator. The flasks were then put in the beakers and sonicated for
different time durations. The absorbance was read at 508 nm.
2.4.3.3. Heating for different times at different heating temperature
(45, 70℃):
2 ml of solution A was transfered to each of three 10 ml volumetric
flasks. This was followed by adding 1 ml of 5 M HCL, 1 ml of 562 µg/ml
ceric ammonium sulfate and 3 ml distilled water to each of the flasks. The
contents of the flasks were heated for different time durations (5,10,15
min). After the time allocated for heating was completed , 1 ml methyl
orange (50µg/ml) was added to each of the three flasks and the volume
made up to the mark using distilled water.
28
Two 100 ml beakers were filled with about 40 ml water and left on the
mantle till the thermometer read 70℃ (±3) after which the flasks were
heated for the specified time durations. A blank was prepared for each of
the 3 samples under the same conditions omitting the addition of the
standard. Absorbance was read at 508 nm.
The same steps mentioned above were followed and the effect of heating
at 45℃ for different time durations was tested at (5, 10, 15, 15, 20, 25,
and 30 min).
2.4.3.4. Acid volume:
The effect of acid volume was tested by transferring 2 ml of solution A
into each of eight 10-ml calibrated volumetric flasks. Different volumes
of 5M HCL (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4) ml were transferred into the
volumetric flasks followed by adding 1 ml of 562 µg/ml ceric ammonium
sulfate to each flask. Using a shaker, the contents of the volumetric
flasks were shaken at a rate 400 osc/min for 15 min.1 ml of 50 µg/ml
methyl orange was added to the contents of each flask and the volume
was made up to the mark with distilled water. A blank was prepared
under the same conditions omitting the addition of the standard.
Absorbance was measured at 508 nm.
2.4.3.5. Shaker rate and time:
The optimum rate was determined by transferring 2 ml of solution A into
each of six 10-ml calibrated volumetric flasks. 1 ml of 5 M HCL and
adding 1 ml of 562 µg/ml ceric ammonium sulfate were then transferred
to each flask. This was followed by using the shaker for shaking the
contents of the flasks for 15 min at different rates 300, 400, 500, 600,
700, 800) osc/min. having completed the shaking process, 1 ml of 50
µg/ml methyl orange was added to each flask and the volume made up to
29
the mark with distilled water. A blank was prepared under the same
conditions omitting the addition of the standard. Absorbance was
measured at 508 nm.
Above procedure was repeated by shaking solutions at 800 osc/min for
different time durations (5, 10, 15, 20, 25, 30, 35 min) to determine the
optimum shaking time.
2.4.3.6. Effect of cerric concentration:
The optimum ceric concentration was determined by transferring 2 ml of
solution A to each of four 10-ml calibrated volumetric flasks followed by
adding 2 ml of 5 M HCL and 1 ml of (450, 562, 675, 900 μg/ml) ceric
ammonium sulfate to each flask. The contents of the flasks were then
shaken at rate of 800 osc/min for 15 min using the shaker followed by
adding 1 ml of 50 µg/ml methyl orange to each flask and making the
volume up to the mark with distilled water.
A blank was prepared under the same conditions omitting the addition of
the standard. Absorbance was read at 508nm.
2.4.4. Validation:
The method validity was investigated in accordance to ICH guidelines,
where linearity, range, detection limit, quantitation limit, accuracy and
precision were determined using the optimum conditions.
2.4.4.1. Linearity:
Calibration series were prepared by micropipetting 0.5, 1, 1.5, 2, 2.5, 3,
3.5) ml of solution A into seven 10-ml volumetric flask. This was
followed by adding 2 ml of 5 M HCL and 3 ml distilled water to each of
the volumetric flasks and shaking their contents at a rate of 800 osc/min
30
for 15 min using a shaker. After completing the shaking process, 1 ml
methyl orange (50 µg/ml) was added to each flask and the volume was
made up to the mark with distilled water. A blank was prepared under the
same conditions omitting the addition of the standard. Absorbance was
read at 508 nm.
Calibrated curve was constructed by plotting concentration against the
absorbance values; this procedure was repeated three times and the
regression data was calculated for the mean calibration curve using excel
sheet.
The sample curve was constructed following the same procedure under
standard curve.
2.4.4.2. Limit of detection:
It is interpreted from the calibration curve of the standard according to
equation (12).
2.4.4.3. Limit of quantification:
It is interpreted from the calibration curve of the STD according to
equation (14).
2.4.4.4. Accuracy:
2.4.4.4.1. Spiked recovery method:
1 ml sample solution C was transferred into three 10 ml volumetric flasks
containing (0.5, 1, 1.5 ml) of standard solution A. This was followed by
adding 2 ml of 5 M HCL and 3 ml distilled water to each flask. The
contents of the flasks were then shaked at rate of 800 osc/min for 15 min
using the shaker. After completing the time allocated for shaking, 1 ml
31
methyl orange (50 µg/ml) was added to each flask and the volume was
made up to the mark with distilled water.
The absorbance values for solutions were recorded at 508 nm against
blank.
The procedure was repeated three times and the mean recovery percent
was allocated at three levels 50, 100 and 150%.
2.4.4.5. Precision:
2.4.4.5.1. Intra-day and Inter-day:
1,2,3 ml of the standard solution A were transferred into three 10 ml
volumetric flasks using micropipette. This was followed by adding 2 ml
of 5 M HCL and 3 ml distilled water to each flask. The contents of the
flasks were then exposed to shaking at rate of 800 osc/min for 15 min
using the shaker. After completing the time allocated for shaking, 1 ml
methyl orange (50 µg/ml) was added to each flask and the volume was
made up to the mark with distilled water.
A blank was prepared under the same conditions omitting the addition of
the standard. Absorbance was measured at 508 nm.
The procedure was repeated three times in the same day for each volume
then the SD and RSD% were calculated using equation (7) and (8)
respectively.
The same steps above were repeated between three days. And the SD and
RSD% were calculated using quotation (7) and (8) respectively.
32
2.4.4.6. Comparison between the developed and official method:
2.4.4.6.1. Single point assay:
Into three 10-ml volumetric flasks 2 ml of the standard stock solution
were transferred using micropipette. This was followed by addition of 2
ml 5 M HCL and 3 ml distilled water to each flask. The contents of the
flasks were then exposed to shaking at rate of 800 osc/min for 15 min.1
ml methyl orange (50 µg/ml) was added to each flask and the volume was
made up to the mark with distilled water.
Absorbance was measured at 508 nm against blank.
The same procedure was done for the sample stock solution instead of the
standard stock solution.
Then the %recovery and RSD% were calculated according to equation
(11) and (8) respectively.
2.4.4.6.2. Official method:
Twenty tablets of PPH were weighed and powdered. A quantity of
powder containing 20 mg of the PPH was then transferred to a 100-ml
volumetric flask followed by adding 20 ml distilled water and shaking the
flask for 10 min. 20 ml of methanol was added to the flask and the
contents were shaken for another 10 min. After completing the shaking
time, sufficient methanol was added to complete the volume to the mark
and filtered. 10 ml of the filtrated solution was diluted to 50 ml using
methanol. The blank was prepared by the same steps and volumes just
without using the drug. The absorbance was measured at 290nm. The
concentration was calculated taking the value of A1% as 206, using
equation (1).
33
2.4.4.6.3. t-test for accuracy:
The value of t was calculated using the equation (10).
2.4.4.6.4. F-test for precision:
The value of F was calculated using the equation (9).
2.4.4.7. Robustness:
The robustness of the method was tested by measuring the sample
absorbance at different wavelengths.
Preparation of the sample involved placing 2 ml of the standard stock
solution into each of five 10-ml calibrated volumetric flasks followed by
addind 1 ml HCL (5 M) and 1 ml ceric ammonium sulfate (562 µg/ml) to
each flask, the contents of the flasks were then shaken at a rate of 800
osc/min for 15 minutes using a shaker. After adding 1 ml methyl orange
(50 µg/ml) to each flask, the volume was completed to the mark with
distilled water.
Without using a standard solution, the same procedure mentioned above
was followed for preparing a blank for each solution. Absorbance was
read at 508 nm.
2.4.4.8. Stability:
The stability of the solution color was determined by measuring the
sample absorbance up to 2 hours.
Both the sample and blank were prepared using the same procedure
mentioned above in 2.4.4.
34
2.4.8. Determination of the molar ratio between the reactants:
2.4.8.1. Determination of molar ratio between the ceric(IV) and the
drug using molar ratio method:
The method was conducted by preparing a series of eleven 10-ml
calibrated volumetric flasks to which different volumes of solution B
(0.888×10-3 M) were added. The volumes were (0, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1) ml. 1 ml of (0.888×10-3 M) ceric ammonium sulfate
was added to each flask followed by shaking the contents of the flasks at
a rate of 800osc/min for 20 minutes using the shaker. After completing
the shaking process, 1 ml methyl orange (50 µg/ml) was added to each
flask and the volume was made up to the mark using distilled water.
A blank was prepared under the same conditions omitting the addition of
the standard. Absorbance was measured at 508 nm.
2.4.8.2. Determination of the molar ratio between ceric (IV) and
methyl orange:
2.4.8.2.1. Using job's method:
A set of eleven 10-ml calibrated volumetric flasks containing different
volumes of ceric ammonium sulfate and methyl orange with the same
molarity (0.222×10-3 M) were prepared. The volumes of ceric ammonium
sulfate transferred into the flasks were (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1) ml. Then different volumes of methyl orange solution (1, 0.9,
0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0) ml were added to the series of
volumetric flasks respectively. Followed by the addition of 2 ml HCL (5
M) to each flask. Using a shaker at a rate of 800 osc/min. the content of
the volumetric flasks were shaken for 20 min. The time allocated for
shaking having been completed, 1 ml methyl orange (50 µg/ml) was
35
added to each flask and the volume was made up to the mark with
distilled water.
A blank was prepared under the same conditions omitting the addition of
the standard. Absorbance was red at 508 nm.
2.4.8.2.2. Using molar ratio method:
A series of fifteen 10-ml calibrated volumetric flasks containg different
volumes of ceric (IV) (0.222×10-3 M) were prepared.
The volumes of ceric transferred into the flasks were (0, 0.5, 1, 1.5, 2,
2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7) ml. 1 ml methyl orange (0.222×10-3 M)
was added to each flask. The blank was 2 ml of 5M HCL and distilled
water. Absorbance was read at 508 nm.
Chapter three
Results
36
Chapter three:
3. Results:
The results of this work are shown in (Tables 4 - 19 and Figures 3 - 16).
3.1 Development of the spectrophotometric method:
3.1.1. Establishment of the maximum wave length:
The maximum absorbance wave length was observed at 508 nm (Figure
1).
Figure 3: Shows scanning of the colored product versus the blank.
508
-0.100
-0.050
0.000
0.050
0.100
0.150
0.200
0.250
200 300 400 500 600 700 800
Ab
sorb
ance
Wave length nm
37
3.2. Method optimization (one factor at a time):
3.2.1. Shaking manually for 15min:
3.2.1.1. The effect of different manual shaking time duration:
Table 4: Shows the effect of different manual shaking time duration on
the absorbance.
Absorbance Time (min)
0.009 0
0.212 5
0.276 10
0.303 15
0.303 20
0.301 25
0.300 30
3.2.1.1. Linearity for the standard:
Tale 5: Illustrates the mean absorbance of seven calibration curves when
manual shaking for 15 min was used as a catalyst.
Mean absorbance Standard concentration
(μg/ml)
0.049 0.5
0.139 1
0.193 1.5
0.307 2
0.375 2.5
0.457 3
38
Figure 4: Demonstrates the mean calibration curve of the standard.
3.2.2. Sonication time:
Figure 5: Shows the absorbance of the standard when the sonicator was
used as a catalyst.
y = 0.1635x - 0.0329R² = 0.9948
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 0.5 1 1.5 2 2.5 3 3.5
Ab
sorb
ance
Concentration µg/ml
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 10 20 30 40 50
Ab
sorb
ance
Sonication time
39
3.2.3. Effect of ceric concentration:
Figure 6: Shows the effect of different ceric concentration when the
sonicator used for 15 min and 2ml of HCL (5 M) was used.
3.2.4. Heating:
:C for different time durations0 Heating at 70 ..4.13.2
Figure 7: Shows the absorbance of the standard when heating at 700 C
was used as a catalyst.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.01 0.02 0.03 0.04 0.05
Ab
sorb
ace
cocentration
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 2 4 6 8 10 12 14 16
Ab
sorb
ance
Concentration
40
3.2.4.2. Heating at 450 C for different time durations:
Figure 8: Shows the absorbance of the standard when heating at 450 C
used as a catalyst.
3.2.5. Acid volume:
Figure 9: Illustrates the effect of different acid volume while using the
shaker for 15 min at rate 400 osc/min.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 5 10 15 20 25 30 35
Ab
sorb
ann
ce
Time
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 1 2 3 4 5
Ab
sorb
ace
Acid volume
41
3.2.6. Shaking time and rate:
3.2.6.1. Effect of different shaking rate:
Figure 10: Shows the effect of shaking by the shaker at different rate.
3.2.6.2. Effect of different shaking time durations at rate 800 osc/min:
Figure 11: illustrates the effect of time durations when the solution was
shaken at rate 800 osc/min.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 200 400 600 800 1000
Ab
sorb
ance
Rate
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 5 10 15 20 25 30 35 40
Ab
sorb
ace
Time
42
3.3. Method Validation:
3.3.1. Construction of the calibration curves:
3.3.1.1. Standard calibration curve:
Table 6: Demonstrates serial dilutions for the mean standard calibration
curve (n= 3).
Mean absorbance of standard Concentration
(μg/ml)
0.073 0.5
0.165 1.0
0.256 1.5
0.344 2.0
0.427 2.5
0.514 3.0
0.616 3.5
Figure 12: Illustrates mean standard calibration curve ( mean absorbance
values, n = 3) .
y = 0.1784x - 0.0147R² = 0.9995
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.5 1 1.5 2 2.5 3 3.5 4
Ab
sorb
ance
Concentration µg/ml
43
3.3.1.2. Sample calibration curve:
Table 7: Shows serial dilutions for the mean sample calibration curve (n
= 3).
Absorbance of standard Concentration
(μg/ml)
0.069 0.5
0.159 1.0
0.252 1.5
0.330 2.0
0.422 2.5
0.511 3.0
0.598 3.5
Figure 13: Shows sample calibration curve.
Using the slope of the standard and the sample calibration curves:
The ratio between them = 0.175
0.178 = 0.98
y = 0.1758x - 0.0171R² = 0.9998
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.5 1 1.5 2 2.5 3 3.5 4
Ab
sorb
ance
Concetration µg/ml
44
3.3.2. LOD and LOQ:
3.3.2.1. Limit of detection (LOD):
Residual standard deviation calculated from the linearity calibration curve
using equation (13).
Residual standard deviation = 4.8508 × 10-3
LOD was calculated using equation (12).
LOD = 3.3×4.8508 × 10−3
0.178 = 0.0899 μg/ml
3.3.2.2. Limit of quantification (LOQ):
LOD was calculated using equation (14).
LOQ = 10×4.8508 × 10−3
0.178 = 0.2725 μg/ml
3.3.3. Regression and validation parameter:
Table 8: Shows regression and validation parameters from the mean
standard calibration curve.
Value Parameter
0.99983 2R
Y =0.178 x- 0.014 Regression equation
0.178 Slope
-0.014 Intercept
356.86 A1%cm
0.5-3.5 Range of linearity (μg/ml)
0.0899 LOD (μg/ml)
0.2725 LOQ (μg/ml)
45
3.3.5. Accuracy:
3.3.5.1. Spiked recovery method:
Table 9: Illustrates recovery percent from standard addition method.
RSD
%
Mean %
Recovery
Abs. of
the
mixture
Abs. of
the
standard
Abs. of the
sample
Conc.
(μg/ml)
3.09 95.56 93.24 0.245 0.075 0.162 50%
(0.5μg/ml) 94.56 0.247 0.078 0.160
98.88 0.251 0.075 0.163
1.417 97.42 97.23 0.338 0.167 0.162 100%
(1μg/ml) 96.15 0.335 0.168 0.160
98.89 0.342 0.167 0.163
0.470 96.42 95.90 0.421 0.256 0.162 150%
(1.5μg/ml) 96.70 0.424 0.259 0.160
96.67 0.427 0.256 0.163
46
3.3.6. Precision:
3.3.6.1. Intra-day:
Table 10: Illustrates intra-day precision calculation data.
RSD% Mean Percent
%
Practical
Conc.
(μg/ml)
A Conc.
(μg/ml)
0.843 102.07 101.13 1.011 0.165 1
102.26 1.023 0.167
102.82 1.028 0.168
0.560 100.85 100.28 2.006 0.341 2
100.85 2.017 0.343
101.41 2.028 0.345
0.606 100.12 99.43 2.983 0.514 3
100.56 3.017 0.520
100.38 3.011 0.519
47
3.3.6.2. Inter-day:
Table11: Demonstrates inter-day precision calculation data.
RSD
%
Mean Percent% Practical
Conc.
(μg/ml)
Absorbance Conc.
(μg/ml)
Day
3
Day
2
Day
1
Day
3
Day
2
Day
1
Day
3
Day
2
Day
1
1.713 100.7
5
98.8
7
102.
26
101.
13
0.98
9
1.023 1.011 0.161 0.167 0.165 1
0.580 100.9
4
101.
41
101.
13
100.
28
2.02
8
2.023 2.006 0.345 0.344 0.341 2
1.093 101 100.
19
100.
56
102.
26
3.00
6
3.017 2.983 0.518 0.520 0.529 3
48
3.3.7. Comparison between the developed and the official method:
3.3.7.1. Single point assay:
Table 12: Demonstrates the percentage of the sample by the developed
method.
RSD% Mean Percent
%
Abs. of
the
standard
Abs. of
the
sample
Conc.
( μg/ml)
Experiment
NO.
0.7499 98.64% 98.54 0.343 0.338 2 1
99.42 0.343 0.341 2 2
97.95 0.342 0.335 2 3
3.3.7.2. Official method:
Table 13: Shows the percentage of the sample by the official method.
RSD
%
Mean Percent% Practical
Conc.
( μg/ml)
Absorbance
Theoretical
Conc.
( μg/ml)
Experiment
NO.
0.140 98.83 98.91 39.563 0.815 40 1
98.67 39.467 0.813 40 2
98.91 39.593 0.815 40 3
49
3.3.7.3. t-test for accuracy:
Table 14: Shows calculation of the mean and RSD for the developed and
the official methods.
Mean
±
RSD
Percent
%
Amount
found by
assay of the
developed
method(mg)
Mean±
RSD
Percent
%
Amount
found by
assay of the
official
method(mg)
Stated
amount
(mg)
Exp. NO.
98.64
±
0.749
98.54 39.42 98.83
±
0.14
98.91 39.56 40 1
99.42 39.77 98.67 39.47 40 2
97.95 39.18 98.91 39.59 40 3
The t value was then calculated using equation (10).
0.3747 =calculated t
4.30 =tabulated t
3.3.7.4. F- test for precision:
The F tabulated was calculated by applying the data from table (7) in
equation (9).
Fcalculated = (0.7398)²
(0.1385)² =
0.5473
0.01918 = 28.535
Ftabulated = 19
50
3.3.8. Robustness:
Table 15: Shows the change in the absorbance in response to small
change in the wave length.
Absorbance WL (nm)
0.332 506
0.329 507
0.336 508
0.328 509
0.327 510
3.3.9. Stability:
Table 16: Illustrates the stability of the color solution with time.
Absorbance Time (min)
0.340 5
0.340 10
0.339 15
0.338 20
0.336 30
0.336 45
0.334 60
0.330 120
51
3.3.10. Determination of the molar ratio between the reactants:
3.3.10.1. Determination of molar ratio between the ceric(IV) and the
drug using Molar Ratio method:
Table 17: Shows the stoichometry between the ceric(IV) and the drug
using molar Ratio method.
A Volume
of the
standard
0 0
0.415 0.1
0.663 0.2
0.670 0.3
0.657 0.4
0.666 0.5
0.676 0.6
0.675 0.7
0.676 0.8
0.675 0.9
0.676 1
52
Figure 14: Illustrates the plot of the molar ratio between ceric(IV) and
the drug using molar ratio method.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 0.2 0.4 0.6 0.8 1 1.2
An
bso
rban
ce
Volume of the standard (ml)
53
3.3.10.2. Determination of the molar ratio between cerric (IV) and
methyl orange:
3.3.10.2.1. Using Job's method:
Table 18: Shows the stoichiometry between ceric (IV) and methyl orange
using Job's method.
A Volume of
methyl
orange
Volume of
cerric (IV)
0.908 1 0
0.836 0.9 0.1
0.721 0.8 0.2
0.612 0.7 0.3
0.518 0.6 0.4
0.411 0.5 0.5
0.305 0.4 0.6
0.181 0.3 0.7
0.091 0.2 0.8
0 0.1 0.9
0 0 1
54
Figure 15: Illustrates the stoichiometry between ceric (IV) and methyl
orange using Job's method.
-0.2
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1 1.2
Ab
sorb
ance
Volume of cerric (ml)
55
3.3.10.2.2. Using molar ratio method:
Table 19: Shows the stoichiometry between ceric (IV) and methyl orange
using molar ratio method.
A Volume
of
ceric(IV)
0.916 0
0.841 0.5
0.693 1
0.596 1.5
0.495 2
0.376 2.5
0.290 3
0.239 3.5
0.113 4
0.067 4.5
0.025 5
0.020 5.5
0.023 6
0.030 6.5
0.033 7
56
Figure 16: Illustrates the stoichiometry between ceric (IV) and methyl
orange using molar ratio method.
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8
Ab
sorb
ance
volume of cerric (ml)
Chapter four
Discussion, conclusion and
recommendations
57
Chapter four:
4.1 Discussion:
Colorimetric analysis is based on the variation of the color of a system in
response to the change in concentration of some constituent.
Many compounds are not themselves colored but can be made to absorb
light in visible region by reaction with suitable reagent. These reactions
are very specific and in most cases very sensitive and the absorbance is
measured at longer wavelengths where the interference from excipients is
far less than at shorter wavelengths.
Due to the pharmacological and therapeutic relevance of PPH, several
methods have been developed for its analysis. Colorimetric method,
particularly which is based on cerium (IV) sulfate spectrophtometry, has
been established as being very sensitive for PPH analysis. The sensitivity
of the method coupled with the fact that cerium (IV) does not seem to
have been fully exploited were the main factors that triggered the desire
to carry out this study.
The proposed method was applied for the determination of PPH in bulk
and tablet dosage form, and is based on the determination of the residual
cerium (IV) sulfate after its reaction with the drug. In acidic medium is
used to ensure the completion of the reaction, and the amount of oxidant
reacted corresponds to the amount of each drug.
The ability of cerium (IV) sulphate to cause oxidation of PPH and bleach
the colour of methyl orange dye has been used for the indirect
spectrophotometric assay of PPH. In this method, PPH reacted with a
measured excess of cerium (IV) sulphate in acidic medium and the
unreacted oxidant was determined by reacting with methyl orange (MO)
followed by absorbance measurement at 508 nm (wavelength of methyl
orange = 465nm in aqueous medium, 510 nm in acidic medium) (Scheme
58
2). The absorbance increased linearly with increasing concentration of the
drug. When increasing amounts of the drug were added to a fixed amount
of cerium (IV) sulphate, the latter was consumed resulting in a
concomitant fall in its concentration. And when fixed amount of the
methyl orange dye was added to decreasing amounts of oxidant, a
concomitant increase in the concentration of dye resulted. This was
observed as a proportional increase in absorbance at the respective λmax
with increasing concentration of the drug (Scheme 1,2 and 3).
The initial experimental conditions were first adopted from the already
established method for the determination of lansoprazol in
pharmaceuticals [51] in which lansoprazol was oxidized by known
excess ceric ammonium sulfate (CAS) . The remaining unreacted CAS
was then reacted with two dyes ( methyl orange and indigo carmine) and
the absorbance was read at the wavelengths 520 nm and 610 nm
respectively. Since PPH has a functional group that can be oxidized by
CAS,it can undergo the same reaction.
59
Scheme 1: The probable oxidation reaction of lansoprazol.
The probable schemes for PPH reactions involved two steps, the first
step of the reaction pathway was proposed by Kudige et al[13] (scheme 2),
and the protonation of methyl orange in acidic media and it's oxidation in
step 2 were proposed by John and Peter[18] (scheme 3 and 4)
60
PPH Oxidized PPH
Scheme 2: Illustrates the reaction between the propanolol and the excess
Ce(IV).
Scheme 3: Demonstrates the species formed on protonation of methyl
orangein acidic medium.[18]
61
Scheme 4: Illustrates the oxidation products of methyle orange.
Thus the whole reaction scheme will be:
Step 1:
PPH Oxidized PPH
62
Step 2:
Scheme 5: Illustrates the reaction scheme for the formation of the
measured color.
63
Each factor was then optimized to obtain the best conditions that result in
the maximum color for the assay of PPH. The concentrations of the drug
and methyl orange were kept the same as that in the method developed by
Kanakapura et al. [51] The concentration of the ceric (IV) 450µg/ml was
found to be so high that it consumed all the drug and methyl orange in the
samples and the absorbance was 0 (Figure 8) (Table 21) and both blank
and sample were colorless (Figure 17). Consequently the concentration
was reduced gradually and then the best one that yielded absorbance
around 4 in the middle of the calibration curve was determined (Table
21).
Findings of the study revealed that regardless of the amount added of the
drug, methyl orange was almost entirely bleached when the addition
order of the reagents was dye+drug+oxidant or dye+oxidant+drug. This
was attributed to the fact that the Ce(IV) sulfate did not have enough time
for oxidizing the drug since it rapidly bleaches methyle orange.
Accordingly the drug and oxidant solution must be added first. However,
no matter which one of the two chemicals (drug and ceric) was added
first, has been shown to have no influence on the reaction. Methyl orange
on the other hand, should be added after a given period of time to allow
for the total oxidation of the drug by Ce(SO4)2. These results were found
to be consistent with those obtained by Abdalla et al. [52] and Iulia et al.
[53] Sara Abdulgader [54]
The study revealed that if methyl orange was immediately added to the
solution containing the drug and Ce(IV) sulfate in acidic medium, the
resultant solution was rapidly bleached yielding a very low absorbance.
This can be explained by the fact that the drug oxidation by cerium(IV) is
a time-developing reaction, thus giving a strong hint for the need of
64
studying the influence of time on the reaction. This finding was consistent
with that of the researches carried out by Abdalla et al. [52] and Iulia et al.
[53] Sara Abdulgader [54] .
Taking into consideration the influence of time on the reaction, enough
time was allowed for quantitative reactions between the drug and cerium
(IV) sulfate to take place accompanied with shaking for different time
durations before adding the indicator and measuring the absorbance. It
was observed that the absorbance of these solutions increased with time
up to 10 minutes after which it remains constant (table 13) .Thus, for
further measurements a reaction time of 15 minutes was selected as the
10 min was considered as a critical time.
The standing time of 5 min was necessary for the bleaching of dye colour
by the residual oxidant which was found to be consistent with the results
obtained by Abdalla et al. [52].
The method was found to be linear in range of 0.5-3 μg/ml (Figure 6)
(Table 14). Nevertheless, linearity was not good which was attributed to
two main reasons. Firstly the reaction was still incomplete and secondly
the inability to maintain the exact shaking rate when performed manually
resulted in subsequent change of absorbance. This necessitated the need
for increasing the catalyst and/or optimizing the shaking.
Using the sonicatior for shaking, it was observed that the absorbance
increased with time then remained steady for about 20 minutes after
which it manifested gradual decrease (Figure 7) (Table 20). A plausible
explanation that can be given for this absorbance pattern is that the
shaking effect provided by the sonicator served in the beginning as a
catalyst for the oxidation reaction. However, as the time increased, the
sonicator started to develop some heat due to the then ongoing shaking
process thus inevitably raising the temperature degree of the sample
65
solution and disrupting the stability of the reaction media leading to
decrease in absorbance.
The findings of the study showed clearly the effect of heating on the
reaction medium and its adverse impact on absorbance. Increase in
heating temperature and time resulted in absorbance decrease which
became drastically sharp at 70℃ and after 15min absorbance reading
became negative (Figure 9, 10) (Table 22, 23). It was also notable that the
color intensity of both the blank and the sample increased with heating
and time till they assumed the same color (pink) (Figure 18, 19, 20).
This can be explained by the fact that ceric solution is unstable at
temperatures above 40℃, and hence it underwent appreciable loss in the
blank. Accordingly when methyl orange was added there was little or
almost no ceric to react with it and hence the color was intense and the
blank absorbed high light. Negative absorbance reading was obviously
attributed to the blank having absorbed more light than the sample. The
ceric instability was demonstrated by Grant [55] whose results revealed
that autoreduction of cerium (IV) sulfate took place in aqueous H2SO4 in
the presence of a glass surface that acted as a catalyst. Similar findings
were reported by Daivid et al [56]. The results were also consistent with
those of Adegoke and Balogun [57] and Abdalla et al [52]who developed
and applied methods based on ceric oxidation for drugs in acidic media
and reported that the temperature made the reaction medium unstable.
Therefore, shaking alone as a catalyst at room temperature 25±5℃ was
adopted.
It is worth mentioning that literature embraces two contradictory views
concerning ceric stability on heating. Sayanna and Venkateshwarlu for
instance claimed that the solution is stable on heating at 60 ± 2°C for 5
minutes in a water bath[58]. However, the majority of researches
66
encountered tend to favor ceric instability on heating which proved quite
consistent with the findings of this study.
The increase in acid volume was met by an increase in the absorbance
which then remained stable from 1.5 ml to 3 ml of the acid followed by
its decline. This result can be explained by the nature of ceric sulfate
reactions in acidic media (Figure 11) (Table 24). These findings were
consistent with those obtained by Adegoke and Balogun [57]. As 1.5 ml
indicated the beginning of the peak, it was considered as critical volume.
The best acid volume was determined as 2 ml 5 M HCL.
Results of the study revealed that when using the shaking machine
(Figure 21) as the shaking rate increase, absorbance increased
accordingly. The optimum shaking rate was found to be the highest rate
800osc/min (Figure 12) (Table 25).
Regarding the shaking time, it was noted that the absorbance increased
with time then remained constant after 10 minutes which was considered
as the critical time. The optimum shaking time duration was 15 min
which was adopted for further measurements (Figure 13) (Table 23).
The oxidation of alcohols by Ce (IV) is believed to proceed by
disproportionation of coordination of complexes. According to the
complex mechanism, unimolecular disproportionation of complex
yields cerrous ion, a proton and a free radical on the alcohol
substrate[59] thus considering the reaction as a complexation reaction. In
addition results obtained by Adegoke and Balogun [57] demonstrated the
relevance of molar ratio and Job's method for calculating the molar ratio
between the reactants. Accordingly a detailed investigation of the two
methods was carried out to evaluate their appropriacy for determining the
molar ratio between the reactants involved in this study (Table 15, 16, 17)
(Figure 14, 15, 16). The results yielded by the Job method were quite
disappointing and the method failed to determine the molar ratio in the
67
type of reaction covered by this research revealing complete
inconsistence with Adegoke and Balogun [57] findings. The failure of
Job's method can be attributed to the fact that the limited range of
proportions presents some rigidity making it difficult to determine the
appropriate molar ratio.
As concerning the molar ratio method, it was found that the ratio
between thw drug and ceric (IV) in step (1) is 1:5 and that between the
ceric (IV) and methyl orange in step (2) is 5:1which was consistence
with results obtained by Adegoke and Balogun. [57]
The developed color remained stable for more than 2 hours. This was
confirmed by measuring the absorbance after certain time intervals (Table
19). This finding was consistent with the result reported by Abdalla et al
[52] and Adegoke and Balogun [57]
It was observed that a small change in the wavelength did not affect the
absorbance values which indicated the robustness of the method (Table
18).
By scanning PPH solution between 200 and 700 nm, the maximum
absorbance wavelength of the solution was found to be 508 nm (Figure
3).
Visual and numerical evaluation of the results obtained from the
calibration curve (Figure 4) confirmed the linearity of the method.
Visual inspection reflected the linearity within the selected range (Table
3, 4) (Figure 4, 5). It was clearly observed that the blank was colorless
while the color intensity of the serial dilutions of the samples increased
from pale to deep pink in the highest concentration (Figure 22). In
addition, values of correlation coefficient R2 was found to be 0.999 for
the standard and the sample, respectively, clearly supporting the observed
68
linearity (Table 6), which is consistent with the ICH guidelines [60]. The
slope of the curve indicated that the method is very sensitive (Table 6).
The ratio obtained from dividing the slope of the sample calibration curve
by the slope of the standard calibration curve was found to be 0.98 which
gave a good indication that the validation parameter would be within the
acceptable ranges.
Limit of detection and quntitation were found to be 0.0899 μg/ml 0.2725
μg/ml respectively (Table 6). It was noticed that both the LOD and LOQ
were less than the smallest concentration in the linear range and this can
be attributed to the high sensitivity of the method. These results were
consistent with those obtained by Kanakapura et al. [51].
Validity evaluated by conduction of accuracy test on bulk and dosage
forms demonstrated that the value of t calculated by the comparison
between the developed and the official method was smaller than the
tabulated one (4.30) which in turn reflected the accuracy of the method
(Table 8, 9, 10).
On the other hand, the results of spiked added recovery indicated good
accuracy according to ICH guidelines which recommend the average
percentage recovery range to be (95.00% – 105.00%)[61] [62](Table 7).
Regarding the evaluation of the inter-day and intra-day variations, the
obtained RSD% values were smaller than 2% reflecting the precision and
robustness of the method within the selected range (Table 11, 12).
All the objectives of the work were successfully performed and were
found to be satisfied.
69
4.2. Conclusions:
A new useful method for the determination of PPH has been developed
and validated as per the current ICH guidelines. The proposed method is
simple, rapid and based on the measurement of stable coloured species
using ceric ammonium sulfate solution as a quantitative reagent.The
proposed method dose not take more than 15–20 min and is among the
most sensitive ever reported for PPH. An additional advantage of the
method is that the absorbance is measured at longer wavelengths where
the interference from excipients is far less than at shorter wavelengths.
70
4.3. Recommendation:
it is recommended to conduct further optimization of the
spectrophotomeric technique that involve ceric (IV) sulfate as an
oxidizing reagent by utilizing experimental design to make use of the
nemours advantages which it offers including its ability to provide more
precise information from a fewer number of experiments and considering
all the multiple variables simultaneously thus demonstrating how the
system works as a whole and enabling the experimenter to optimize the
critical responses.
Experimental design must be fully exploited to distinguish between the
main factors and the less important ones, to determine the best reaction
conditions, to make a final decision on heating effect and to determine
whether the nature of the drug itself plays an influential role in the
reaction media or not.
Ceric (IV) sulfate must be further investigated with respect to its stability
and the effect of heat on the reactions in which it takes part as a reagent
so that optimization of the experimental conditions can be based on
concrete scientific evidence rather than relying on trial and error.
The method is recommended for routine use in pharmaceutical
laboratories as a part of industrial quality control.
The method is recommended for routine use in pharmaceutical
laboratories as a part of industrial quality control.
References
71
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Appendices:
Table20: Illustrates the absorbance of the standard when the sonicator
was used as a catalyst.
Absorbance Time
(min)
0.299 5
0.350 10
0.361 15
0.372 20
0.380 25
0.288 30
0.273 35
0.251 40
Table 21: Shows the effect of different cerric concentration.
Absorbance Cerric concentration (g/50ml 5M
H2SO4)
0.627 0.0225
0.381 0.0281
0.246 0.03375
0.000 0.045
The sonicator used for 15 min with 2ml of 5 M HCL.
79
C0 heating at 70e absorbance of the STD when : Demonstrates thTable 22
was used as a catalyst.
Absorbance Time (min)
0.120 5
0.020 10
-0.012 15
C 0 heating at 45 e absorbance of the STD when: demonstrates thTable 23
was used as a catalyst.
Absorbance Time
(min)
0.328 5
0.345 10
0.353 15
0.316 20
0.266 25
0.156 30
80
Table 24: Illustrates the effect of different acid volume on the absorbance
while using the shaker for 15 min at rate 400 osc/min.
Absorbance Acid volume
(ml)
0.181 0.5
0.204 1
0.282 1.5
0.288 2
0.285 2.5
0.282 3
0.196 3.5
0.105 4
Table 25: Shows the effect of shaking by the shaker on the absorbance at
different rates.
Absorbance Rate
(osc/min)
0.221 300
0.288 400
0.286 500
0.333 600
0.339 700
0.346 800
81
Table 26: Illustrates time effect on the absorbance at shaking rate of 800
osc/min.
Absorbance Time (min)
0.260 5
0.340 10
0.343 15
0.341 20
0.344 25
0.344 30
0.342 35
Figure 17: Illustrates the difference in color manifested by the sample
and blank at ceric concentration of 900µg/ml.
82
Figure 18: Illustrates the difference in color between the sample and
blank when heating at 45℃ for 20, 25 and 30min was used as a catalyst.
Figure 19: Illustrates the differences in color between the sample and
blank when heating at 70℃ for 5 and 10 min was used as a catalyst.
83
Figure 20: Illustrates the differences in color between the sample and
blank when heating at 70℃ for 5 and 15 min was used as a catalyst.
Figure 21: Demonstrates the shaking machine (Shaker).
84
Figure 22: Demonstrates the gradual increase in the color intensity in
response to increased sample concentration.